Immunomodulatory compositions and methods of use thereof

ABSTRACT

The present disclosure provides immunomodulatory compositions comprising heat-killed  Caulobacter crescentus  (HKCC). Immunomodulatory compositions of the present disclosure are useful for modulating an immune response in an individual. The present disclosure thus provides methods of modulating an immune response in an individual, involving administering an immunomodulatory composition comprising HKCC to the individual.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/108,744 filed Jun. 28, 2016, which is a national phase filing under35 U.S.C. § 371 of International Application No. PCT/IB2015/050108 filedJan. 7, 2015, which claims the benefit of U.S. Provisional PatentApplication No. 61/924,607, filed Jan. 7, 2014, each of which isincorporated herein by reference in its entirety.

INTRODUCTION

Caulobacter crescentus is non-pathogenic, harmless, aquatic,gram-negative bacterium that grows at ˜23° C. in many soil andfreshwater environments Caulobacter has been studied for nearly 50years. The main laboratory strain (C. crescentus CB15) is wellcharacterized genetically and biochemically, and the genome of C.crescentus has been sequenced. Caulobacters are readily grown usingstandard laboratory equipment. They can also be easily grown incommercial fermenters to at least 30 ODs in animal protein free, definedminimal media.

There is a need in the art for safe and effective vaccines that induceboth cellular and humoral immune responses. There is a need in the artfor immunomodulatory compositions and adjuvants that can be used totreat infections, cancers, and autoimmune diseases.

SUMMARY

The present disclosure provides immunomodulatory compositions comprisingheat-killed Caulobacter crescentus (HKCC). Immunomodulatory compositionsof the present disclosure are useful for modulating an immune responsein an individual. The present disclosure thus provides methods ofmodulating an immune response in an individual, involving administeringan immunomodulatory composition comprising HKCC to the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the effect of heat-killed Caulobacter crescentus (HKCC)as a mucosal adjuvant to induce T cell responses against OVA. HKCC at50×10⁶ CFU/mouse induces higher antigen specific T cell responsesfollowing single intranasal immunization of C57/b16 male mice with amixture of OVA antigen (50 μg/mouse) than lower doses (0.5-5×10⁶CFU/mouse). Mice were euthanized 2 wks after immunization. Values arethe mean of triplicates with ±SD.

FIGS. 2A-E illustrate the effect of live Caulobacter crescentus (CC) andHKCC on antigen-specific cellular immune responses against OVA. Groupsof five C57/b16 mice were immunized with live CC or HKCC at 50×10⁶CFU/mouse with OVA (20 μg/mouse) in 100 μl total volume/mouse by thesubcutaneous (s.c.) route at the base of the tail on days 0 and 14. Micewere euthanized 2 wks after immunization. Values are the mean oftriplicates with ±SD. HKCC stimulates robust cell mediated (CD4, CD8)immunity against chicken ovalbumin (OVA) antigen as compared to live CC.2A. T cell proliferative response from spleen; 2B. T cell proliferativeresponse from lymph nodes and 2C. GrB producing antigen specific CTLs.2C: Frequency of GrB-producing cells, by ELISPOT, two weeks after s.c.immunization with OVA. 2D: spleen T cell proliferation after a singles.c. injection of OVA. 2E: Serum IgG (left panel) and serum IgGa) rightpanel, one week after a single s.c. immunization.

FIGS. 3A-L depicts the effect of HKCC as an adjuvant for therapeutic HBVvaccine to induce cellular and humoral immune responses. Antigenspecific T cell (CD4+, CD8+) and antibody responses following twointranasal immunizations (at 14 day intervals) of C57/b16 male mice witha mixture of recombinant HBV core antigen (5 μg/mouse) and HKCC (50×10⁶CFU/mouse). Values are the mean of triplicates with ±SD. 3A. HBV coreantigen specific T cell proliferation one week after two immunizations;3B. HBV core antigen specific T cell proliferation three weeks after twoimmunizations; 3C. IFN-gamma production; 3D. IL-12 production; 3E.IFN-gamma production; 3F. Number of GrB producing T cell spots; 3G. HBVcore antigen specific serum IgG responses; 3H. HBV core antigen specificserum IgG2a responses; 3I. HBV core antigen specific lung IgG responsesone wk after two immunizations; 3J. HBV core antigen specific lung IgAresponses one wk after two immunizations; 3K. HBV core antigen specificlung IgG responses three wk after two immunizations; 3L. HBV coreantigen specific lung IgA responses three wk after two immunizations.

FIG. 4 illustrates that HKCC exhibits potent adjuvant activity enhancingHCV derived NS3 specific T cell responses. C57Bl/6 female mice wereimmunized twice (at 10 day intervals) with a mixture of 10 lipopeptides(NS3 1248-71, 1621-40, 1127-46, 1187-1206, 1367-86, 1487-1506, 1507-26,1547-66, 1607-26, 1637-57, 2.5 μg each peptide/mouse) and HKCC (50×10⁶CFU/mouse) s.c. The mice were euthanized 15 days after secondimmunization. HCV antigen specific T cell responses of spleen fromimmunized vs. unimmunized mice are shown. Values are the mean oftriplicates with ±SD.

FIGS. 5A-C depict the effect of HKCC as an adjuvant for tuberculosisvaccine and leads to reduction of mycobacterial load in lungs, liver andspleen. Mice were immunized twice subcutaneously (at 12 days intervals)with a mixture of 7 monolipopeptides (Ag85B 68-88, 93-112, 126-142,143-167, 199-218, 240-251, 257-273, 5 μg each peptide/mouse) and HKCC(50×10⁶ CFU/mouse). The immunized mice were challenged with 0.5×10⁶cfu/mouse Mycobacterium tuberculosis (Mtb) H37Ra six weeks after secondimmunization. Infected mice were euthanized three weeks after Mtbchallenge. Lungs, liver and spleen were collected from individual miceand used for CFU assay. The CFU data are shown for five individual micein each experimental group in 5A. lungs, 5B. liver, and 5C. spleen.

FIGS. 6A-D depict the effect of HKCC as an adjuvant for prophylacticvaccine for solid tumor to reduce EL-4 tumors after a singlesubcutaneous (s.c.) immunization. Groups of five C57Bl6 mice wereimmunized once subcutaneously with a mixture of irradiated EL-4 cells(1×10⁶/mouse) and HKCC (50×10⁶ CFU/mouse). The immunized mice werechallenged with 0.25×10⁶ EL-4 cells/mouse in 100 μl PBS s.c. in thelower left flank eight days after immunization. Tumor growth wasmeasured for 28 days after challenge using digital calipers in twoperpendicular directions, and mice were humanely euthanized. Tumor areawere calculated as length×width (in mm). The data represent 6A. tumorprogression; 6B. tumor mass; 6C. EL-4 specific lymph node T cellproliferation response and 6D. EL-4 specific serum IgG response. Tumordata are shown for five individual mice in each experimental group andimmune responses are from five pooled mice.

FIGS. 7A-C depict the effect of HKCC as an adjuvant for prophylacticvaccine for lung cancer to reduce in lung metastases after a single s.c.immunization. Groups of five C57Bl6 mice were immunized oncesubcutaneously with a mixture of irradiated B16 cells (1×10⁶/mouse) andHKCC (50×10⁶ CFU/mouse). The immunized mice were challenged with 0.4×10⁶B16 cells/mouse in 50 μl PBS intravenously in the tail vein eight daysafter immunization. Mice were humanely euthanized 12 days after tumorchallenge. The data represent A. lung tumor nodules in both treated anduntreated groups; B. lungs weight; and C. B16 cell lysate specific serumIgG response. Tumor data are shown for five individual mice in eachexperimental group and immune responses are from five pooled mice.

FIGS. 8A-C illustrate antitumor activity of HKCC against B16 melanomalung metastasis after two s.c. treatments. Groups of four C57Bl6 micewere challenged with 0.4×10⁶ B16 cells/mouse in 100 μl PBS intravenouslyin the tail vein. Starting from day 3 post tumor challenge, HKCC (50×10⁶cfu/mouse) was administered s.c. once weekly for a total of two weeks.Three days after the last treatment, mice were euthanized. The datarepresent 8A. lung tumor nodules in both treated and untreated groups;8B. lung weights and 8C. B16 cell lysate specific serum IgG response.Tumor data are shown for four individual mice in each experimental groupand lung weights represent Avg±SD from four mice.

FIGS. 9A-B depict efficacy of immunotherapeutic treatment with HKCC inmice challenged with EL-4 tumor cells. Groups of five C57Bl6 mice werechallenged with 0.25×10⁶ EL-4 cells/mouse in 100 μl PBS s.c. in thelower left flank. Six days after tumor challenge, mice were treated onceweekly subcutaneously with HKCC (50×10⁶ CFU/mouse) or PBS control threetimes. Tumor growth was measured for 28 days after challenge usingdigital calipers in two perpendicular directions, and mice were humanelyeuthanized. Tumor area were calculated as length×width (in mm). The datarepresent: 9A, tumor progression; 9B, PD-1 expression on immune cells inspleens. Tumor data shown represent mean from five mice in eachexperimental group and PD-1 data are from five pooled spleens from mice.

FIGS. 10A and 10B illustrate the effect of live CC and HKCC onantigen-specific cellular (CD4⁺ and CD8⁺ T cells) immune responsesagainst TIV (seasonal) influenza vaccine upon single mucosal (i.n.)immunization with a low dose of antigen. Groups of five C57/b16 micewere immunized by the intranasal route with live CC or HKCC at 50×10⁶CFU/mouse with Vaxigrip (1.6 μg/mouse) in 30 μl total volume/mouse. Inthe control no adjuvant group, Vaxigrip (1.8 μg/mouse) alone wasadministered subcutaneously. Mice were euthanized 8 days afterimmunization. Values are the mean of triplicates with ±SD. The datarepresent 10A. antigen specific T cell proliferation and 10B. antigenspecific GrB producing CTLs.

FIGS. 11A-G depict the effect of HKCC in inducing long-lastingantigen-specific humoral and cellular immune responses againstco-administration of multiple antigens of influenza upon mucosal (i.n.)immunizations with low doses of antigens. Antigen specific T cell andantibody responses were determined following one or two intranasalimmunization(s) (at 21 days interval) of C57/b16 male mice with amixture of seasonal TIV influenza vaccine (Vaxigrip 1.8 Kg/mouse),M2e-monolipo peptide (20 μg/mouse) and HKCC (50×10⁶ CFU/mouse). HKCCwhen combined with other adjuvants e.g., MPL (a TLR-4 agonist) (5μg/mouse) or a polymeric compound e.g., poly-L-arginine hydrochloride(100 μg/mouse), potentiates immune responses. No IgE were developedagainst antigens at both early and later time points. Values are themean of triplicates with ±SD. 11A. Vaxigrip and M2e antigens specific Tcell proliferation 8 days after two immunizations; 11B. anti-Vaxigripantibody responses in lung lavage; 11C. anti-M2e antibodies in lunglavage; 11D. anti-Vaxigrip antibody responses in nasal lavage; 11E.anti-M2e antibodies in nasal lavage; 11F. anti-Vaxigrip antibodyresponses in serum; 11G. anti-M2e antibodies in serum.

FIG. 12 depicts the effect of HKCC on activating TLRs and NLRs mediatedsignaling. The activity of HKCC (at concentrations 10⁴-10⁸ cfu/ml) onvarious TLRs and NLRs was assessed with human embryonic kidney cells(HEK 293) expressing individual TLR or NLR (Invivogen) using thesecretory embryonic alkaline phosphatase (SEAP) reporter gene that islinked to NF-kB activation in response to TLR or NLR stimulation. TheSEAP activity was measured using Quanti-blue substrate.

FIG. 13 depicts the effect of S-layer negative HKCC on antigen-specificT cell response against multiple antigens of influenza upon mucosal(i.n.) immunizations. Antigen specific T cell responses followingintranasal immunizations of C57/b16 male mice with a mixture of seasonalTIV influenza vaccine (Vaxigrip 1.8 μg/mouse), M2e-monolipo peptide (20pig/mouse) and S-layer negative HKCC (50×10⁶ CFU/mouse). Values are themean of triplicates. Vaxigrip and M2e antigens specific T cellproliferation 8 days after two immunizations (at 21 days interval).

FIGS. 14A-D depict enhancement of spectrum of protection of seasonal fluTIV vaccine (Vaxigrip) upon single mucosal or s.c. immunization fromheterologous virus infection. Groups of three BALB/c female mice wereimmunized by the intranasal (1.8 μg/mouse Vaxigrip) or s.c. (3.6μg/mouse Vaxigrip) routes with HKCC (50×10⁶ CFU/mouse) in 30 and 100 μltotal volume/mouse, respectively. In the control no adjuvant group,Vaxigrip (3.6 μg/mouse) alone was administered subcutaneously. Eightdays after immunization, mice were challenged intranasally with 30μl/mouse of stock of H1N1 (PR8) virus and daily weights of individualmouse were recorded. Four days after infection, mice were euthanized andviral titers were determined in lung homogenates. BAL was collected todetermine cytokine and infiltrating cells. The data demonstrates 14A.viral titers in the lungs of infected mice; 14B. % weight loss afterinfection; 14C. production of cytokine IL-2 in BAL after infection; and14D. infiltration of various innate and adaptive immune cells in BAL ofthe immunized and unimmunized groups.

FIGS. 15A and 15B depict the effect of a single subcutaneousimmunization of a poorly immunogenic antigen (M2e) adjuvanted withWT-HKCC or LPS-negative HKCC on weight-loss after influenza virusinfection. Groups of five BALB/c female mice were immunizedsubcutaneously with M2e peptide or lipopeptide (25 μg/mouse) and HKCC orLPS-negative HKCC (50×10⁶ CFU/mouse) in 100 μl total volume/mouse at thebase of the tail. Eight days after immunization, mice were challengedintranasally with 30 μl/mouse of stock of H1N1 (PR8) virus and dailyweights of individual mouse were recorded. Four days after infection,mice were euthanized and BAL was collected to determine infiltratingcells. The data demonstrates 15A. percent weight loss after infection;and 15B. infiltration of various innate and adaptive immune cells in BALof the immunized and unimmunized groups.

FIGS. 16A-D depict the effect of prophylactic immunotherapy with HKCC bys.c., i.n., or oral route(s) on protection from viral (H1N1) infection.Groups of five BALB/c female mice were treated with HKCC (50×10⁶cfu/mouse) by s.c. (100 μl volume/mouse at the base of the tail), i.n.(30 μl total/mouse) or oral route in (100 μl volume/mouse). Twenty fourhours after treatment, mice were challenged intranasally with 30μl/mouse of stock of H1N1 (PR8) virus and daily weights of individualmouse were recorded. Two and five days after infection, mice wereeuthanized and BAL samples and lungs were collected. The datademonstrates 16A. % weight loss after infection; 16B. viral titers inlungs; 16C. infiltration of various innate and adaptive immune cells inBAL and 16D. cytokines present in BAL.

FIG. 17 depicts immunotherapeutic antiviral effect of HKCC against H1N1infection. Groups of five BALB/c female mice were challengedintranasally with 30 μl/mouse of stock of H1N1 (PR8). Twenty-four hoursafter the infection, mice were treated with HKCC (50×10⁶ cfu/mouse)subcutaneously (100 μl volume/mouse at the base of the tail),intranasally (30 μl total/mouse) or orally (100 μl volume/mouse). Fivedays after infection, mice were euthanized and lungs were collected todetermine viral titers. The data demonstrate viral titers in lungs ofindividual mice and the average of five mice.

FIGS. 18A and 18B depict inhibition of HCV 1a RNA replication bysupernatants of human PBMCs stimulated with HKCC. Human PBMCs from asingle donor #1 were stimulated with 10⁵, 10⁶ or 10⁷ HKCC/ml (Wild Type(HK WT CC), LPS negative (HK LPS-CC) and S layer negative (HK Slayer-CC) (FIG. 18A), followed by collecting the supernatant at 24 hrs.The supernatant (500 μl/ml) was then added to the HCV1a repliconcontaining Huh-7 cells, incubated for 5 days and cells were examined forHCV RNA copy numbers by real-time RT-PCR (FIG. 18A) Inhibition of HCV 1aRNA replication upon single treatment with supernatants (300 μl and 450μl) of human PBMCs stimulated with 10⁷ HKCC/ml with or withouttelaprevir (Tela) or ribavirin (RB V) and incubation for 5 days,followed by HCV RNA quantification by real-time RT-PCR (FIG. 18B).PolyI:C stimulated PBMC media was used as controls. M stands for mediacontrol (untreated cells) and PBMCs or PBS represents supernatant fromunstimulated PBMCs. The data represent means of triplicates.

FIG. 19 demonstrates that HKCC induces cytokines from human PBMCs whichcan inhibit intracellular bacterial replication. Human monocytic cellline (THP-1) was infected with M. avium or Mtb H37Ra using publishedprocedures, followed by two treatments (on days 0 and 4) withsupernatants (50%) collected from human PBMCs treated for 24 hrs withHKCC or PBS in 24 well plates. Supernatants collected from threedifferent donor PBMCs stimulated with HKCC were tested as donors #1, #2and #3. In controls clarithromycin or rifampicin were added directly toinfected THP-1 cells. Five days after second treatment, THP-1 werecollected, lysed and plated on 7H11 agar plates to determine bacterialCFUs.

FIG. 20 illustrates the effect of HKCC in combination with achemotherapeutic drug in reducing bacterial burden. Groups of 5 BALB/cfemale mice were challenged with H37Ra (0.5×10⁶ cfu/mouse)intravenously. Five days post infection, mice were treated with HKCC(50×10⁶ cfu/mouse) and INH (20 μg/mouse), INH alone or PBS using aschedule shown in the figure. Mice were euthanized 2 days after the lasttreatment. Spleens, lungs and liver were collected to determinebacterial loads using CFU assay.

FIG. 21 demonstrates that HKCC enhances T cell responses againstmalaria-derived antigen Spf66. A group of five C57/b16 male mice wereimmunized subcutaneously twice (at 12 days interval) with HKCC(50×10⁶/mouse)+Spf66 peptide (20 μg/mouse), Spf66 peptide (20 μg/mouse)alone or PBS. Mice were euthanized eight days after second immunization.The data represent malaria antigen (Spf66)-specific T cell proliferationfrom splenocytes; the values are the mean of triplicates ±SD.

FIGS. 22A-C demonstrate that HKCC induces antigen-specific T cellresponses upon oral immunization and viral challenge. Groups of fiveBALB/c female mice were immunized twice orally (at 12 days interval)with M2e lipopeptide (50 μg/mouse)+HKCC (50×10⁶ CFU/mouse), M2elipopeptide (50 μg/mouse) alone or PBS in 200 μl total volume/mouse.Twelve days after immunizations, mice were challenged intranasally with30 μl/mouse of stock of H1N1 (PR8) virus. Four days after infection,mice were euthanized. Spleens and BALs were collected. Antigen specificT cell proliferation (22A), activation of CTLs in splenocytes (22B) andinfiltration of activated CTLs in BALs (22C) are shown.

FIG. 23 demonstrates cellular immune responses generated against HCV NS3and a pool of 15-aa long peptides from HCV-NS3 in mice immunized withadenoviral vector (rAd-NS3) in the absence or presence of HKCC. Groupsof five C57bl/6 female mice were immunized twice (at 14 days interval)with 2×10⁷ pfu/mouse adeno vector expressing NS3 coding region (rAd-NS3)with or without HKCC (50×10⁶ cfu/mouse) intramuscularly (i.m.) inquadriceps muscles in a total volume of 150 microlitre/mouse.PBS-immunized mice were used as negative control. Eight days aftersecond immunization, mice were euthanized and spleens were collected.Enriched T cells (4×10⁵/well) from spleens were cultured with irradiatedsyngeneic spleen cells as APCs (4×10⁵/well) and recombinant HCV NS3protein or HCV NS3 derived synthetic peptides pool (5 μg/m′) for fourdays. Proliferation of T cells was examined by ³H thymidineincorporation assay, and stimulation indices were calculated using theformula (SI=CPMs in the presence of antigen/CPMs in the absence ofantigen). All data represent mean±standard deviations of triplicatewells.

FIG. 24 demonstrates that HKCC mixed with IFA elicits strong T cellresponses following single subcutaneous immunization with a low dose ofantigen (Vaxigrip) and challenge with heterologous (H1N1) influenzavirus. Groups of five BALB/c mice were immunized by the subcutaneousroute with HKCC at 50×10⁶ CFU/mouse, IFA (20 ul) with Vaxigrip (0.5μg/mouse) in 100 μl total volume/mouse. In the control no adjuvantgroup, Vaxigrip (0.5 μg/mouse) alone was administered subcutaneously.Eight days after immunization, mice were challenged with H1N1 influenzavirus, and euthanized three days after infection. The data representantigen specific T cell proliferation from splenocytes and inguinallymph nodes. Values are the mean of triplicates with ±SD.

FIG. 25 depicts that recombinant HKCC containing hemagglutinin proteinfrom influenza virus (H5-HKCC) after intranasal immunization inducesinfluenza antigens' specific T proliferative responses. Groups of fiveBALB/c female mice were immunized with recombinant H5-HKCC or wild-typeHKCC (50×10⁶ cfu/ml) twice intranasally (at 8 days interval) andchallenged with H1N1 influenza 12 days after second immunization. Micewere euthanized 3 days after infection. The data represent influenzaantigens (vaxigrip) specific T cell proliferation from splenocytes andthe values are the mean of triplicates with ±SD.

FIGS. 26A and 26B illustrate the effect of live CC and/or HKCC onantigen-specific humoral immune responses against multiple antigens ofinfluenza upon single s.c. immunization and heterologous influenza viruschallenge. Groups of five BALB/s female mice were immunized with amixture of seasonal TTV influenza vaccine (Vaxigrip 1.0 μg/mouse),M2e-monolipo peptide (20 μg/mouse) and HKCC (50×10⁶ CFU/mouse); Vaxigrip(1.0 μg/mouse), M2e-monolipo peptide (20 μg/mouse) and live CC (50×10⁶CFU/mouse); Vaxigrip (1.0 μg/mouse), M2e-monolipo peptide (20 μg/mouse);or PBS once subcutaneously. Mice were challenged intranasally with H1N1influenza virus eight days after immunization. Sera samples werecollected 4 days after infection (11 days after single immunization) andexamined for antibodies against Vaxigrip (26A) and M2e (26B).

DEFINITIONS

The terms “individual,” “host,” “subject,” and “patient” are usedinterchangeably herein, and refer to mammals, including, but not limitedto, humans, non-human primates (e.g. simians), non-human mammals (e.g.,mammalian livestock animals (e.g., bovine, porcine, caprine, and ovineanimals)), and mammalian pets (e.g., cats, dogs); fish; and birds (e.g.,chicken).

A “biological sample” encompasses a variety of sample types obtainedfrom an individual. The definition encompasses blood, serum, plasma, andother liquid samples of biological origin; solid tissue samples such asa biopsy specimen or tissue cultures or cells derived therefrom and theprogeny thereof. The definition also includes samples that have beenmanipulated in any way after their procurement, such as by treatmentwith reagents; washed; or enrichment for certain cell populations, suchas epithelial cells. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, organs,tissue samples, lung biopsy samples, lung epithelial cells,gastrointestinal epithelial cells, gastrointestinal tract tissuesamples, bronchoalveolar lavage (BAL) fluid samples, nasal lavage fluidsamples, blood, plasma, serum, cerebrospinal fluid, fecal samples, andthe like.

An “immunomodulator” or “immunomodulatory agent” is any agent which doesone or more of: restores depressed immune function, regulates abnormalimmune function, enhances normal immune function, and provide desiredimmune response. Immune function includes one or more of: humoral(antibody-mediated) immunity, cellular immunity, and innate immunity. An“immunomodulalor” includes agents acting directly on the cells involvedin the expression of immune response, or on cellular or molecularmechanisms, which, in turn, act to modify the function of cells involvedin immune response. Augmentation of immune function may result from theaction of an immunomodulatory agent to abrogate suppressive mechanismsderived by negative-feedback influences endogenous or exogenous to theimmune system. Thus, immunomodulators can have diverse mechanisms ofaction.

An “adjuvant” is any agent which is capable of potentiating an immuneresponse and are, therefore, one class of immunopotentiators (Stites andTen, Basic and Clinical Immunology, 7^(th) Ed., Appleton and LangeNorwalk Conn. pp. 797, 1991). Adjuvants are used to increase the immuneresponses in vaccination in order to enhance the humoral and/or cellmediated immune responses.

A “vaccine” is intended to encompass a preventive vaccine or atherapeutic vaccine. A preventive vaccine is one that is given tostimulate an immune response to an antigen, so that if an individualsubsequently is exposed to the antigen, the pre-formed immunity willprotect the individual from the respective disease related to theantigen. A therapeutic vaccine is given to an individual who already hasa disease associated with an antigen, wherein the vaccine can elicit animmune response or boost the individual's existing immunity to theantigen, to treat and/or ameliorate symptoms of the disease.

A “cytokine” means any secreted polypeptide that affects the functionsof other cells, and is a molecule, which modulates interactions betweencells in the immune or inflammatory response. A cytokine includes, butis not limited to monokines, chemokines, and lymphokines, regardless ofwhich cells produce them.

The terms “antibodies” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype, fragments of antibodies which retainspecific binding to antigen, including, but not limited to, Fab, Fv,scFv, and Fd fragments, chimeric antibodies, humanized antibodies,single-chain antibodies, bi-specific antibodies, and fusion proteinscomprising an antigen-binding portion of an antibody and a non-antibodyprotein. Also encompassed by the term are Fab′, Fv, F(ab′)₂, and orother antibody fragments that retain specific binding to antigen, andmonoclonal antibodies. An antibody may be monovalent or bivalent.

A “therapeutically effective amount” or “efficacious amount” means theamount of a compound or agent that, when administered to a mammal orother subject for treating a disease, is sufficient to effect suchtreatment for the disease. The “therapeutically effective amount” willvary depending on the compound or agent, the disease and its severityand the age, weight, general health status, sex, etc., of the subject tobe treated. In some cases, an “effective amount” of an agent is anamount that: 1) restores the immune function to normal levels; 2)increases immune function above normal levels; or 3) reduces immunefunction below a pathological level.

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease or symptom in a mammal, and includes:(a) preventing the disease or symptom from occurring in a subject whichmay be predisposed to acquiring the disease or symptom but has not yetbeen diagnosed as having it; (b) inhibiting the disease or symptom,i.e., arresting its development; or (c) relieving the disease, i.e.,causing regression of the disease. The therapeutic agent may beadministered before, during or after the onset of disease or injury. Thetreatment of ongoing disease, where the treatment stabilizes or reducesthe undesirable clinical symptoms of the patient, is of particularinterest. Such treatment is desirably performed prior to complete lossof function in the affected tissues. The subject therapy will desirablybe administered during the symptomatic stage of the disease, and in somecases after the symptomatic stage of the disease.

A “pharmaceutically acceptable carrier or excipient” means a non-toxicsolid, semi-solid, or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. One skilled in the art of preparingformulations can readily select the proper form and mode ofadministration depending upon the particular characteristics of the gentselected, the disease state to be treated, the stage of the disease, andother relevant circumstances.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “aheat-killed Caulobacter crescentus” includes a plurality of suchheat-killed bacteria and reference to “the adjuvant” includes referenceto one or more adjuvants and equivalents thereof known to those skilledin the art, and so forth. It is further noted that the claims may bedrafted to exclude any optional element. As such, this statement isintended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides immunomodulatory compositions comprisingheat-killed Caulobacter crescentus (HKCC). Immunomodulatory compositionsof the present disclosure are useful for modulating an immune responsein an individual. The present disclosure thus provides methods ofmodulating an immune response in an individual, involving administeringan immunomodulatory composition comprising HKCC to the individual.

Immunomodulatory Compositions

The present disclosure provides immunomodulatory compositions comprisingheat-killed Caulobacter crescentus (HKCC). HKCC in an immunomodulatorycomposition of the present disclosure are non-viable and aremetabolically inactive. An immunomodulatory composition of the presentdisclosure can comprise a cocktail of one or more different strains ofCaulobacter crescentus bacteria.

HKCC-containing immunomodulatory compositions include the HKCC by itselfwith a pharmaceutically acceptable carrier or excipients forimmunological adjuvant activity, including “adjuvanting” in which HKCCadministration to a subject may be wholly independent of, and/orseparated temporally and/or spatially from, administration to thesubject of one or more antigens against which elicitation or enhancementof an immune response (e.g., an antigen specific response) in thesubject is desired.

An immunomodulatory composition of the present disclosure can increasean immune response in an individual. In some cases, an effective amountof an immunomodulatory composition of the present disclosure is anamount that is effective, when administered in a single dose or inmultiple doses, to increase the number of B cells in an individual. Forexample, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase thenumber of B cells in an individual by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold,compared to the number of B cells in the individual in the absence oftreatment with the immunomodulatory composition. In some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number of antigen-specific Bcells in an individual. For example, in some cases, an effective amountof an immunomodulatory composition of the present disclosure is anamount that is effective, when administered in a single dose or inmultiple doses, to increase the number of antigen-specific B cells in anindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, or more than 10-fold, compared to the numberof antigen-specific B cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase activity of B cellsin an individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase activation of B cells in an individual by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least25-fold, at least 50-fold, at least 100-fold, or more than 100-fold,compared to the activation level of B cells in the individual in theabsence of treatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the amount ofantibody specific to a given antigen in the individual. For example, insome cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the amount ofantibody specific to a given antigen in an individual by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, or morethan 10-fold, compared to the amount of antibody specific to the antigenin the individual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase production of one ormore cytokines in the individual. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase production of one or morecytokines in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, or more than 10-fold, compared to theamount of the cytokine in the individual in the absence of treatmentwith the immunomodulatory composition. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase production of GM-CSF in anindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, or more than 10-fold, compared to the amountof GM-CSF in the individual in the absence of treatment with theimmunomodulatory composition. For example, in some cases, an effectiveamount of an immunomodulatory composition of the present disclosure isan amount that is effective, when administered in a single dose or inmultiple doses, to increase production of IL-22 in an individual by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least10-fold, or more than 10-fold, compared to the amount of IL-22 in theindividual in the absence of treatment with the immunomodulatorycomposition. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase production of interferon (IFN)-α and/or IFN-β and/or IFN-γin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, or more than 10-fold, compared to theamount of IFN-α or IFN-β or IFN-γ in the individual in the absence oftreatment with the immunomodulatory composition. As another example, insome cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase production of one ormore of IL-17A, 1L-2, IL-10, IL-6 and TNF-α in all individual by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least10-fold, or more than 10-fold, compared to the amount of IL-17A, IL-2,IL-10, IL-6, or TNF-α in the individual in the absence of treatment withthe immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase a Th1 response in anindividual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase a Th1 response in an individual by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, more than10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 100-fold, or more than 100-fold, compared to the levelof the Th1 response in the individual in the absence of treatment withthe immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of CD4⁺ T cells in an individual. For example, in some cases,an effective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofCD4⁺ T cells in an individual by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least100-fold, or more than 100-fold, compared to the number and/or activityof CD4⁺ T cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD4⁺ T cellsin an individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD4⁺ T cellsin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the number and/or activity ofantigen-specific CD4⁺ T cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of CD8⁺ T cells in an individual. For example, in some cases,an effective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofCD8⁺ T cells in an individual by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least100-fold, or more than 100-fold, compared to the number and/or activityof CD8⁺ T cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD8⁺ T cellsin an individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD8⁺ T cellsin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the number and/or activity ofantigen-specific CD8⁺ T cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of cytolytic T cells in an individual. For example, in somecases, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase the number and/oractivity of cytolytic T cells in an individual by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, more than10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 100-fold, or more than 100-fold, compared to thenumber and/or activity of cytolytic T cells in the individual in theabsence of treatment with the immunomodulatory composition. In somecases, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase the number and/oractivity of antigen-specific cytolytic T cells in an individual. Forexample, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase thenumber and/or activity of antigen-specific cytolytic T cells in anindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity ofantigen-specific cytolytic T cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of one or more of natural killer (NK) cells, NKT cells,macrophages, and dendritic cells (DCs) in an individual. For example, insome cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of one or more of NK cells, NKT cells, macrophages, and DCs inan individual by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity of one or more ofNK cells, NKT cells, macrophages, and DCs in the individual in theabsence of treatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to modulate the number and/oractivity of Tregs in an individual. Tregs (regulatory T cells) are CD4⁺or CD8⁺, and may also be FoxP3⁺. Tregs may also be defined by othermarkers such as PD-1, CTLA-4 etc. Regulatory cells may also be comprisedof other innate cells such as NK, NKT and DCs, and B lymphocytes.“Modulate the number and/or activity” of Tregs, as used herein, refersto increasing, decreasing, or balancing the number and/or activity ofTregs. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to decrease the number and/or activity of Tregs in an individual by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 75%, ormore than 75%, compared to the number and/or activity of Tregs in theindividual in the absence of treatment with the immunomodulatorycomposition. In some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase thenumber and/or activity of Tregs in an individual by at least 10%, atleast 25%, at least 50%, at least 2-fold, at least 5-fold, or at least10-fold, or more than 10-fold, compared number and/or activity of Tregsin the individual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of Th17 cells in an individual. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofTh17 cells in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, more than 10-fold, at least 15-fold,at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold,or more than 100-fold, compared to the number and/or activity of Th17cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th17 cells inan individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th17 cells inan individual by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity ofantigen-specific Th17 cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of Th22 cells in an individual. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofTh22 cells in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, more than 10-fold, at least 15-fold,at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold,or more than 100-fold, compared to the number and/or activity of Th22cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th22 cells inan individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th22 cells inan individual by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity ofantigen-specific Th22 cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to elicit, boost and/or regulateinnate and/or adaptive (including both cellular and humoral) immuneresponses in an individual. For example, in some cases, an effectiveamount of an immunomodulatory composition of the present disclosure isan amount that is effective, when administered in a single dose or inmultiple doses, to modulate (e.g., increase) the number and/or activityof innate and/or adaptive immune cells and/or their effector functionsin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the number and/or activity of one ormore of innate or adaptive immune cells and/or their effector functionsin the individual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to protect innate and/or adaptiveimmune cells from depletion or prevent their apoptosis in an individual.For example, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to protectinnate and/or adaptive immune cells from depletion or prevent theirapoptosis in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, more than 10-fold, at least 15-fold,at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold,or more than 100-fold, compared to the number and/or activity of one ormore of innate or adaptive immune cells and/or their effector functionsin the individual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to induce proliferation and/ordifferentiation of hematopoietic stem cells, and restore homeostasis.For example, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to induceproliferation and/or differentiation of hematopoietic stem cells, andrestore homeostasis in an individual by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, atleast 100-fold, or more than 100-fold, compared to the individual in theabsence of treatment with the immunomodulatory composition.

In some cases, an immunomodulatory composition of the present disclosurecomprises HKCC and an antigen. Where an immunomodulatory composition ofthe present disclosure comprises HKCC and an antigen, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase an immune response to the antigenby at least about 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the immune response to the antigen in theabsence of treatment with the immunomodulatory composition. For example,where the antigen is an antigen associated with or derived from a cancercell, a pathogenic bacterium, a pathogenic virus, or a pathogenicprotozoan, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase an immune response tothe antigen by at least about 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the immune response to the antigen inthe absence of treatment with the immunomodulatory composition. Theimmune response can be a humoral immune response, e.g., a B cell orantibody immune response. Thus, e.g., in some cases, where the antigenis an antigen associated with or derived from a cancer cell, apathogenic bacterium, a pathogenic virus, or a pathogenic protozoan, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase a B cell response to the antigenby at least about 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the B cell response to the antigen in theabsence of treatment with the immunomodulatory composition. For example,in some cases, where the antigen is an antigen associated with orderived from a cancer cell, a pathogenic bacterium, a pathogenic virus,or a pathogenic protozoan, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase theamount of antibody specific to the antigen by at least about 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, more than10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 100-fold, or more than 100-fold, compared to theamount of antibody specific to the antigen in the absence of treatmentwith the immunomodulatory composition. The immune response can be acellular immune response, e.g., a T cell response. Thus, e.g., in somecases, where the antigen is an antigen associated with or derived from acancer cell, a pathogenic bacterium, a pathogenic virus, or a pathogenicprotozoan, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase a T cell response to theantigen by at least about 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the T cell response to the antigen in theabsence of treatment with the immunomodulatory composition. In somecases, the immune response is a humoral immune response and a cellularimmune response.

An immunomodulatory composition of the present disclosure can compriseHKCC in an amount of from about 10³ HKCC per ml to about 10¹² HKCC perml. For example, an immunomodulatory composition of the presentdisclosure can comprise HKCC in an amount of from about 10³ HKCC per mlto about 10⁴ HKCC per ml, from about 10⁴ HKCC per ml to about 10⁵ HKCCper ml, from about 10⁵ HKCC per ml to about 10⁶ HKCC per ml, from about10⁶ HKCC per nil to about 10⁷ HKCC per ml, from about 10⁸ HKCC per nilto about 10⁹ HKCC per ml, from about 10⁹ HKCC per ml to about 10¹⁰ HKCCper ml, from about 10¹⁰ HKCC per ml to about 10¹¹ HKCC per ml, or fromabout 10¹¹ HKCC per ml to about 10¹² HKCC per ml.

An immunomodulatory composition of the present disclosure can compriseHKCC in an amount of from about 10² to about 10²⁰ colony forming units(cfu) per unit dosage form; for example, an immunomodulatory compositionof the present disclosure can comprise HKCC in an amount of from about10² to about 10³ from about 10³ to about 10⁵, from about 10⁵ to about10⁷, from about 10⁷ to about 10⁹, from about 10⁹ to about 10¹¹, fromabout 10¹¹ to about 10¹³, from about 10¹³ to about 10¹⁵, from about 10¹⁵to about 10¹⁸, or from about 10¹⁸ to about 10²⁰, cfu per unit dosageform. A unit dosage form can be an amount that is administered in asingle dose; for example, a unit dosage form can be 0.5 ml, 1.0 ml, orother volume suitable for administration in a single dose.

HKCC can be generated by exposing Caulobacter crescentus to atemperature of from about 37° C. to about 95° C. for a time period offrom about 1 minute to about 2 hours. For example, HKCC can be generatedby exposing Caulobacter crescentus to a temperature of about 60° C. for1 hour. As another example, HKCC can be generated by exposingCaulobacter crescentus to a temperature of 80° C. for about 30 minutes.HKCC are non-viable.

Alternatively, Caulobacter crescentus can be inactivated by chemicaltreatment, e.g., by treating the bacteria with glutaraldehyde orformalin. Alternatively, Caulobacter crescentus can be inactivated byirradiation, e.g., microwave irradiation, gamma irradiation, X rays,ultraviolet or infrared light irradiation, a photochemical processcombining treatment with a synthetic psoralen and long-wave UV light,etc. Alternatively, Caulobacter crescentus can be inactivated by afreeze-thaw method, freeze-drying, sonication, french press sonication,lysis, cryo preservation or any other non-denaturing method. Otherprocesses may be used for the inactivation of Caulobacter crescentusthat are known to those of ordinary skill in the art.

Inactivation of Caulobacter species can be performed by treatment withacidic and/or basic conditions, various aldehydes (e.g., glutaraldehyde,formaldehyde), chemicals (e.g., beta propriolactone), solvents andvarying salt concentrations. Modulating metabolic enzymes is anothermethod of inactivating Caulobacter, which can be achieved by modifyingculture nutrients, limiting or providing excess of various chemicalssuch as nucleoside tri phosphates, carbohydrates, cyclic nucleosidemonophosphates (e.g., 3′,5′-cyclic GMP, 8-Bromo, N2,O2′-dibutyryl cyclicGMP) etc. in the growth medium. Further, metabolic enzymes can bemodulated by genetic engineering, whereby a given enzyme can be eitherknocked in or knocked out from Caulobacter sp. (J S Poindexter, TheCaulobacters: Ubiquitous Unusual Bacteria, Microbiol Rev 45, 123-179,1981).

Inactivation of Caulobacter for use as immunomodulatory agent describedherein can also be achieved by treatment with anti-metabolite orantibiotic agents such as mitomycin C, penicillin G, cisplatin andderivatives etc., DNA cross-linking or methylating agents such asethidium bromide, which can inhibit further replication/division ofbacteria (J S Poindexter, The Caulobacters: Ubiquitous Unusual Bacteria,Microbiol Rev 45, 123-179, 1981).

Antigens

An immunomodulatory composition of the present disclosure can comprise,in addition to HKCC, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more than 10) antigens. Suitable antigens include, but are notlimited to, an antigen derived from a pathogenic microorganism; atumor-associated antigen; and an allergen. Antigens derived from apathogenic microorganism include antigens derived from a virus, abacterium, a fungus, a protozoan, or a helminth.

In some embodiments, Caulobacter crescentus is genetically modified toproduce an antigen; and the genetically modified Caulobacter crescentusis heat-killed, to produce an immunomodulatory composition of thepresent disclosure. Methods of genetically modifying bacteria are knownin the art.

In other embodiments, HKCC is admixed with an antigen in animmunomodulatory composition of the present disclosure. Caulobactercrescentus can act as a carrier and/or delivery vehicle to deliverantigens. As a non genetic modification (GM), such as electrostatic andhydrophobic interactions, binding of antigens to the Caulobactercrescentus surface may enable the Caulobacter crescentus to act as anantigen carrier and/or delivery vehicle. Further, due tobioadhesion/mucoadhesion, Caulobacter crescentus may facilitate antigenuptake by M cell transport, delivery to and subsequentactivation/maturation of DCs/APCs, induction of NK, NKT, B and T cellresponses at mucosal surfaces.

An antigen, for use in certain embodiments of the herein describedimmunomodulatory compositions and methods employing HKCC, may be anytarget epitope, molecule, molecular complex, cell or tissue againstwhich elicitation or enhancement of immunogenicity in a subject isdesired.

An immunomodulatory composition of the present disclosure can includeone or more antigens or antigenic compositions capable of eliciting animmune response against a human or animal pathogen. The antigen can bederived from at least one infectious pathogen that is selected from avirus, a bacterium, a mycobacterium, a mycoplasma, a fungus, a tumor ora cancer cell. In certain embodiments, the antigen may be associatedwith autoimmune disease, allergy, asthma, prion disease or any otherconditions where stimulation of an antigen-specific response would bedesirable or beneficial.

A suitable antigen can be any type of antigen known in the art. Antigenscan be produced in any of a variety of sources such as plants, animals,prokaryotes, in vitro cell culture, etc. Antigens can be in variety offorms as described below.

Suitable antigens include, e.g., peptides, modified peptides, peptidemimotopes, conformationally-constrained synthetic peptides,multi-epitope peptides from one or more antigens, branched peptides,lipopeptides, monolipopeptides, dilipopeptides, peptides conjugated orfused to proteins, peptides conjugated or fused to T cell or B cellepitopes. See, e.g., U.S. Pat. No. 8,198,400. Suitable antigens include,e.g., full-length antigens, truncated antigens, mutated antigens, andinactivated or combined forms from a single pathogen or differentpathogen(s) or cancer. Suitable antigens include, e.g., proteins,purified or recombinant proteins, recombinant fusion proteins, proteinsand peptides conjugated to toll-like receptor (TLR) agonists, proteinsand peptides conjugated to bacterial toxins, proteins and peptidesconjugated to antibodies, proteins and peptides conjugated to cytokinesand chemokines, glycoproteins, glycolipoproteins and derivativesthereof. Suitable antigens include, e.g., polysaccharides,polysaccharide conjugates, oligosaccharides, lipids, glycolipids,carbohydrates and derivatives thereof. Suitable antigens include smallmolecules, e.g., morphine, nicotine and derivatives thereof. An antigencan be modified to enhance antigen presentation and/or co-stimulation,or inhibit co-inhibitory signals. A poorly immunogenic antigen can beconjugated to a carrier such as keyhole limpet hemocyanin (KLH),albumin, hepatitis B virus (HBV) core antigen, etc.

An antigen or antigenic composition can be obtained from live viruses,dead viruses, attenuated viruses, bacteria, fungi, protozoa, helminths,etc.

An antigen can be a whole cell extract, a cell lysates, a whole cell, awhole live cell, a whole inactivated cell, a whole irradiated cell, etc.An antigen can be a whole live, dead, inactivated, irradiated orattenuated pathogenic or non-pathogenic microorganism. Antigens may becrude, purified, or recombinant form. In some cases, an antigen is atleast 50% pure, at least 60% pure, at least 70% pure, at least 80% pure,at least 90% pure, at least 95% pure, at least 98% pure, or at least 99%pure, or more than 99% pure.

An antigen can be chemically, enzymatically, or genetically coupled toHKCC. In some cases, an antigen is present in an immunomodulatorycomposition of the present disclosure in admixture with HKCC.

An immunomodulatory composition of the present disclosure can comprise asingle type of antigen. An immunomodulatory composition of the presentdisclosure can include 2 or more different antigens. An immunomodulatorycomposition of the present disclosure can include 2, 3, 4, 5, 6, or morethan 6, different antigens. Where an immunomodulatory composition of thepresent disclosure includes more than one antigen, the more than oneantigen can be from the same pathogenic organism, or from the samecancer cell. Where an immunomodulatory composition of the presentdisclosure includes more than one antigen, the more than one antigen canbe from two or more different pathogenic organisms, or from two or moredifferent cancer cells or two or more different types of cancers.

An antigen can be in the form of a protein, a lipopolysaccharide, alipoprotein, a proteoglycan, glycoproteins, glycosaminoglycans, anoligosaccharide, etc.

An antigen can be in the form of a nucleic acid comprising a nucleotidesequence encoding an antigen, e.g., a polypeptide antigen. For example,an antigen can be provided in the form of DNA (e.g., plasmid DNA, nakedDNA etc.), RNA, and/or a wild-type, attenuated and/or recombinantvector-based nucleic acid. The nucleic acid coding for the antigen canbe either “naked” or contained in a delivery system, such as liposomes.

A recombinant vector-encoded antigen can be at least one recombinantexpression construct which comprises a promoter operably linked to anucleotide sequence encoding an antigen in recombinant viral vectors(such as adenovirus (e.g. Ad2, Ad4, Ad5, Ad35, Ad35K5 etc.),adeno-associated virus, lentivirus, herpes virus, poxvirus, vesicularstomatitis virus, alpha virus, measles virus, papaya mosaic virus,cytomegalovoirus, modified vaccinia Ankara virus MVA, polio virus, Marbavirus etc.), bacterial vector vaccines (such as Salmonella, Shigella, E.coli, Lactococcus lactic, Listeria sp., Lactobacillus sp.), fungalvectors (such as heat killed recombinant Saccharomyces yeast), plantviruses, virus-like particles (VLPs), virosomes, synthetic vaccineparticles, synthetic biomimetic supramolecular biovectors,depathogenized viral/bacterial strains (such as NIBRG14 from H5N1). Thevector could be in the form of live wild-type, non-replicative, mutated,modified, defective or attenuated. The vectors could be from human,animal, plant or prokaryote origin and in any effective amount.

In treating or preventing infectious disease, cancer or autoimmunediseases, antigen can be given at the same or different times, at thesame or different site than the immunostimulatory composition of thepresent disclosure.

Antigens from Pathogenic Bacteria

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, an antigen derived from or associatedwith a pathogenic bacterium. In some cases, an immunomodulatorycomposition of the present disclosure comprises, in addition to HKCC,one or more bacterial antigens, e.g., 1, 2, 3, 4, 5, or more bacterialantigens, from one or more bacteria.

Non-limiting examples of pathogenic bacteria include Mycobacteria,Streptococcus, Staphylococcus, Pseudomonas, Salmonella, Neisseria, andListeria. In some cases, the bacteria is Neisseria gonorrhea,Mycobacterium tuberculosis (Mtb), M. leprae, M. bovis, M. avium, M.smegmetis, M. paratuberculosis, Listeria monocytogenes, Streptococcuspneumoniae, S. pyogenes, S. agalactiae, S. viridans, S. aureus, S.epidermis, S. faecalis, or S. bovis.

Other examples of bacteria contemplated include, but are not limited to,Gram positive bacteria (e.g., Listeria, Bacillus such as Bacillusanthracis, Erysipelothrix species), Gram negative bacteria (e.g.,Bartonella, Brucella, Burkholderia, Campylobacter, Enterobacter,Escherichia, Francisella, Hemophilus, Klebsiella, Morganella, Proteus,Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, andYersinia species), spirochete bacteria (e.g., Borrelia species includingBorrelia burgdorferi that causes Lyme disease), anaerobic bacteria(e.g., Actinomyces and Clostridium species including C. difficile), Grampositive and negative coccal bacteria, Enterococcus species including E.fetalis, E. faecium, Streptococcus species, Pneumococcus species,Staphylococcus species, Neisseria species.

Additional non-limiting examples of specific infectious bacteria includeCitrobacter, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophila, Mycobacteria avium, M. intracellulare, M. kansaii, M.gordonae, M. africanum, Staphylococcus aureus, Neisseria meningitidis,Haemophilus influenzae, Bacillus anthracis, Y. pestis, Corynebacteriumdiphtheriae, Erysipelothrix rhusiopathiae, Clostridium perfringens,Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae,Pasturella multocida, Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira,Rickettsia, P. gingivalis and Actinomyces israelli.

An antigen can be derived from any of the aforementioned bacteria.

Non-limiting examples of suitable bacterial antigens include pertussistoxin, filamentous hemagglutinin, pertactin, FIM2, FIM3, adenylatecyclase and other pertussis bacterial antigen components; diphtheriabacterial antigens such as diphtheria toxin or toxoid and otherdiphtheria bacterial antigen components; tetanus bacterial antigens suchas tetanus toxin or toxoid and other tetanus bacterial antigencomponents; streptococcal bacterial antigens such as M proteins,adhesins, lipoteichoic acid, pneumonolysins and other streptococcalbacterial antigen components; gram-negative bacilli bacterial antigenssuch as lipopolysaccharides, toxins and other gram-negative bacterialantigen components; Borrelia bacterial antigens such as OspA, OspC, DbPAor DbPB; Mycobacterium tuberculosis bacterial antigens such as mycolicacid, heat shock protein 65 (HSP65), the 30 kDa major secreted protein,ESAT-6, antigen 85A, 85B and 85C, ID83, ID93 and other mycobacterialantigen components; Helicobacter pylori bacterial antigen componentssuch as urease, catalase, vacuolating toxin; pneumococcal bacterialantigens such as pneumolysin, pneumococcal capsular polysaccharides,pneumococcal surface protein A and other pneumococcal bacterial antigencomponents; haemophilus influenza bacterial antigens such as capsularpolysaccharides, adhesins, lipoproteins and other haemophilus influenzabacterial antigen components; anthrax bacterial antigens such as anthraxprotective antigen and other anthrax bacterial antigen components;Nisseria spp. bacterial antigens such as capsular polysaccharides,transferrin-binding proteins, lactoferrin-binding proteins and adhesins,rickettsiae bacterial antigens such as rompA and other rickettsiaebacterial antigen component; Chlamydia bacterial antigens such as Momp,heparin binding proteins, ORF3 and other proteins. Also included withthe bacterial antigens described herein are any other bacterial,mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.

A bacterial antigen can be purified (e.g., at least 50% pure, at least60% pure, at least 70% pure, at least 80% pure, at least 90% pure, atleast 95% pure, at least 98% pure, or at least 99% pure, or more than99% pure). A bacterial antigen can be an extract from a bacterial cell.A bacterial antigen can be synthetically produced, e.g., by recombinantmeans.

Fungal Antigens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, one or more fungal antigens, e.g., 1, 2,3, 4, 5, or more fungal antigens, from one or more fungi.

Fungal antigens suitable for inclusion in an immunomodulatorycomposition of the present disclosure include, but are not limited to,e.g., candida fungal antigen components; histoplasma fungal antigenssuch as heat shock protein 60 (HSP60) and other histoplasma fungalantigen components; cryptococcal fungal antigens such as capsularpolysaccharides and other cryptococcal fungal antigen components;Coccidioides fungal antigens such as spherule antigens and otherCoccidioides fungal antigen components; and tinea fungal antigens suchas trichophytin and other Coccidioides fungal antigen components.

Fungal antigens suitable for inclusion in an immunomodulatorycomposition of the present disclosure can be obtained from Candida spp.including C. albicans, Aspergillus spp., Cryptococcus spp. including C.neoformans, Blastomyces sp., Pneumocytes spp., or Coccidioides spp.

Parasite Antigens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, a parasite antigen. Parasites includeprotozoan parasites and helminths. In some cases, an immunomodulatorycomposition of the present disclosure comprises, in addition to HKCC,one or more parasitic antigens, e.g., 1, 2, 3, 4, 5, or more parasiticantigens, from one or more parasites.

Examples of parasites include Plasmodium spp., Toxoplasma gondii,Babesia spp., Trichinella spiralis, Entamoeba histolytica, Giardialamblia, Enterocytozoon bieneusi, Naegleria, Acanthamoeba, Trypanosomarhodesiense and Trypanosoma gambiense, Isospora spp., Cryptosporidiumspp, Eimeria spp., Neospora spp., Sarcocystis spp., and Schistosoma spp.

Parasite antigens can be derived from Plasmodium spp. (such as RTS, S,TRAP, MSP-1, MSP-3, RAP1, RAP2 etc.), Toxoplasma spp. including T.gondii (such as SAG2, SAG3, Tg34), Entamoeba spp. including E.histolytica, Schistosoma spp., Trypanosoma cruzi Cryptosporidium spp.,Angiostrongylus spp., Ancyclostoma spp., Wuchereria spp., Brugia spp.,Giardia spp., Leishmania spp., Pneumonocystis spp., Enterobius spp.,Ascaris spp., Trichuris spp., Trichomonas spp., Necator spp., Onchocercaspp., Dracanculus spp., Trichinella spp., Strongyloides spp.,Opisthorchis spp., Paragonimus spp., Fasciola spp., or Taenia spp.

Protozoan Antigens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, a protozoan antigen. A protozoan antigencan be derived from any protozoan parasite, including, but not limitedto, Giardia; a plasmodium species (e.g., Plasmodium falciparum);Toxoplasma gondii; a Cryptosporidium; a Trichomonas species; atrypanosome (e.g., Trypanosoma cruzi); or Leishmania.

Protozoan antigens include, but are not limited to, e.g., Plasmodiumfalciparum antigens such as merozoite surface antigens, sporozoitesurface antigens, circumsporozoite antigens, gametocyte/gamete surfaceantigens, blood-stage antigen pf 155/RESA and other plasmodial antigencomponents, and parasites killed by freeze-thawing etc.; toxoplasmaantigens such as SAG-1, p30 and other toxoplasmal antigen components;schistosomae antigens such as glutathione-S-transferase, paramyosin, andother schistosomal antigen components; Leishmania major and otherleishmanial antigens such as gp63, lipophosphoglycan and its associatedprotein and other leishmanial antigen components; and Trypanosoma cruziantigens such as the 75-77 kDa antigen, the 56 kDa antigen and othertrypanosomal antigen components.

Helminth Antigens

Helminth antigens that can be included in an immunomodulatorycomposition of the present disclosure include antigens derived fromflatworms, thorny-headed worms, and roundworms (nematodes).

Viral Antigens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, one or more viral antigens, e.g., 1, 2,3, 4, 5, or more viral antigens, from one or more viruses.

Viruses that can be the source of the viral antigen(s) include, but arenot limited to, herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6,HHV-8), influenza viruses (Flu A, B), hepatitis viruses (HepA, HepB,HepC, HepD, HepE), human immunodeficiency viruses (HIV-1, HIV-2),respiratory syncytial viruses, measles viruses, rhinoviruses,adenoviruses, SARS viruses, papillomaviruses, orthopoxviruses, West Nileviruses, and a dengue viruses. Viruses that can be the source of theviral antigen(s) include members of the Flaviviridae family of viruses.Viruses that can be the source of the viral antigen(s) include aflavivirus selected from the group consisting of dengue, Kunjin,Japanese encephalitits, West Nile, and yellow fever virus. Viruses thatcan be the source of the viral antigen(s) include lymphocyticchoriomenignitis virus, hepatitis B virus, Epstein Barr virus, and humanimmunodeficiency virus. Viruses that can be the source of the viralantigen(s) include, but are not limited to: Retroviridae (e.g. humanimmunodeficiency viruses, such as HIV-1, also referred to as LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.strains that cause gastroenteritis); Togaviridae (e.g. equineencephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses,encephalitis viruses, yellow fever viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola-like viruses, Marburg viruses);Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus,respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses);Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae(e.g. reoviruses, orbiviruses and rotaviruses); Bornaviridae;Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (e.g.,adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2),varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae(variola viruses, vaccinia viruses, pox viruses); Bunyaviridae (e.g.,Rift valley fever virus, Schmallenberg virus); and Iridoviridae (e.g.African swine fever virus); and unclassified viruses (e.g. theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis, thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1, internally transmitted;class 2, parenterally transmitted, i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses.

Suitable viral antigens include antigens from the herpesvirus family,including proteins derived from herpes simplex virus (HSV) types 1 and2, such as HSV-1 and HSV-2 glycoproteins gB, gC, gD, gE, gH and ICP27;antigens derived from varicella zoster virus (VZV) such as gpI, II,IE-63, Epstein-Barr virus (EBV) such as gp350 and cytomegalovirus (CMV)including CMV gB and gH; and antigens derived from other humanherpesviruses such as HHV6 and HHV7. (See, e.g. Chee et al.,Cytomegaloviruses (J. K. McDougall, ed., Springer-Verlag 1990) pp.125-169, for a review of the protein coding content of cytomegalovirus;McGeoch et al., J. Gen. Virol. (1988) 69:1531-1574, for a discussion ofthe various HSV-1 encoded proteins; U.S. Pat. No. 5,171,568 for adiscussion of HSV-1 and HSV-2 gB and gD proteins and the genes encodingtherefor; Baer et al., Nature (1984) 310:207-211, for the identificationof protein coding sequences in an EBV genome; and Davison and Scott, J.Gen. Virol. (1986) 67:1759-1816, for a review of VZV.)

Suitable viral antigens include antigens from the hepatitis family ofviruses, including hepatitis A virus (HAV), hepatitis B virus (HBV),hepatitis C virus (HCV), the delta hepatitis virus (HDV), hepatitis Evirus (HEV) and hepatitis G virus (HGV), can also be conveniently usedin the techniques described herein. By way of example, the viral genomicsequence of HCV is known, as are methods for obtaining the sequence.See, e.g., International Publication Nos. WO 89/04669; WO 90/11089; andWO 90/14436. The HCV genome encodes several viral proteins, including E1(also known as E) and E2 (also known as E2/NSI) and an N-terminalnucleocapsid protein (termed “core”) (see, Houghton et al., Hepatology(1991) 14:381-388, for a discussion of HCV proteins, including E1 andE2). Each of these proteins, as well as antigenic fragments thereof,will find use in the present composition and methods.

Suitable viral antigens include the 6-antigen from HDV (see, e.g., U.S.Pat. No. 5,378,814). Additionally, antigens derived from HBV, such asthe core antigen, the surface antigen, sAg, as well as the presurfacesequences, pre-S1 and pre-S2 (formerly called pre-S), as well ascombinations of the above, such as sAg/pre-S1, sAg/pre-S2,sAg/pre-S1/pre-S2, and pre-S1/pre-S2, are suitable. See, e.g., “HBVVaccines—from the laboratory to license: a case study” in Mackett, M.and Williamson, J. D., Human Vaccines and Vaccination, pp. 159-176, fora discussion of HBV structure; and U.S. Pat. Nos. 4,722,840, 5,098,704,5,324,513; Beames et al., J. Virol. (1995) 69:6833-6838, Birnbaum etal., J. Virol. (1990) 64:3319-3330; and Zhou et al., J. Virol. (1991)65:5457-5464.

Suitable viral antigens include, but are not limited to, proteins frommembers of the families Picornaviridae (e.g., polioviruses, etc.);Caliciviridae; Togaviridae (e.g., rubella virus, dengue virus, etc.);Flaviviridae (e.g., nucleoprotein, VP35, VP40, glycoprotein, L protein);Coronaviridae; Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabiesvirus, etc.); Filoviridae; Paramyxoviridae (e.g., mumps virus, measlesvirus, respiratory syncytial virus, etc.); Orthomyxoviridae (e.g.,influenza virus types A, B and C, etc.); Bunyaviridae; Arenaviridae(e.g., arenaviruses, tick-fever viruses); Retroviradae (e.g., HTLV-I;HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.)),including but not limited to antigens from the isolates HIV-IIIb,HIV-SF2, HIV-LAV, HIV-LAI, HIV-MN); HIV-1-CM235, HIV-1-US4; HIV-2;simian immunodeficiency virus (SIV) among others. Additionally, antigensmay also be derived from human papillomavirus (e.g., HPV6; HPV11, HPV16;HPV18) such as E1, E2, E5, E6, E7, L1, L2 proteins and the tick-borneencephalitis viruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed.1988); Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe,eds. 1991), for a description of these and other viruses.

Suitable viral antigens include the gp120 or gp140 envelope proteinsfrom any of the above HIV isolates, including members of the variousgenetic subtypes of HIV, are known and reported (see, e.g., Myers etal., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, N.Mex. (1992); Myers et al., Human Retroviruses and Aids, 1990, LosAlamos, N. Mex.: Los Alamos National Laboratory; and Modrow et al., J.Virol. (1987) 61:570-578, for a comparison of the envelope sequences ofa variety of HIV isolates) and antigens derived from any of theseisolates will find use in the present methods. Suitable viral antigensinclude proteins derived from any of the various HIV isolates, includingany of the various envelope proteins such as gp160 and gp41, gagantigens such as p24gag and p55gag, as well as proteins derived from thepol, nef, and tat regions, as well as core regions.

Suitable viral antigens include antigens of influenza virus.Specifically, the envelope glycoproteins HA and NA of influenza A can beused. Numerous HA subtypes of influenza A have been identified (Kawaokaet al., Virology (1990) 179:759-767; Webster et al., “Antigenicvariation among type A influenza viruses,” p. 127-168. In: P. Palese andD. W. Kingsbury (ed.), Genetics of influenza viruses. Springer-Verlag,New York). Conserved antigens of influenza such as nucleoprotein, M2 andM1 can also be used in vaccine compositions. Thus, proteins derived fromany of these isolates can also be used in the compositions and methodsdescribed herein.

Suitable viral antigens include antigens of respiratory syncytial virussuch as F, N, M, G proteins. Suitable viral antigens include antigens ofDengue virus such as NS1, NS3, and NS5 proteins. Thus, proteins derivedfrom any of these isolates can also be used in the compositions andmethods described herein. Suitable viral antigens for non-human mammalsand other animals include, but are not limited to, antigens of porcineepidemic diarrhea (PED) virus, Foot and mouth diseases virus, classicalswine fever virus, rabies virus, Pseudorabies virus, infectious bovinerhinotracheitis (IBR) virus, avian influenza, West Nile virus, chickeninfectious anemia virus, bovine viral diarrhea virus (BVDV), equineherpes viruses, simian immunodeficiency virus, feline leukemia virus,feline sarcoma virus etc.

Cancer-Associated Antigens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, a cancer-associated antigen.Cancer-associated antigens can be derived from the cell surface,cytoplasm, nucleus, organelles and the like of cells of tumor tissue.Cancer associated antigens can also be associated with tumor-supportmechanisms e.g., angiogenesis and tumor invasion. Tumor associatedantigens (TAAs) may be autologous tumor cells (e.g., irradiated,sonicated, lysed, etc.). In some cases, an immunomodulatory compositionof the present disclosure comprises, in addition to HKCC, one or morecancer antigens, e.g., 1, 2, 3, 4, 5, or more cancer antigens, from oneor more cancers.

Examples of cancer-associated antigens include, without limitation,antigens associated with hematological cancers such as leukemias andlymphomas, neurological tumors such as astrocytomas or glioblastomas,melanoma, breast cancer, lung cancer, head and neck cancer,gastrointestinal tumors such as gastric or colon cancer, liver cancer,pancreatic cancer, genitourinary tumors such cervix, uterus, ovariancancer, vaginal cancer, testicular cancer, prostate cancer or penilecancer, bone tumors, vascular tumors, or cancers of the lip,nasopharynx, pharynx and oral cavity, esophagus, rectum, gall bladder,biliary tree, larynx, lung and bronchus, bladder, kidney, brain andother parts of the nervous system, thyroid, Hodgkin's disease,non-Hodgkin's lymphoma, multiple myeloma and leukemia.

Cancer-associated antigens include, e.g., mutated oncogenes; viralproteins associated with tumors; and tumor mucins and glycolipids. Theantigens may be viral proteins associated with tumors. Certain antigensmay be characteristic of tumors (one subset being proteins not usuallyexpressed by a tumor precursor cell), or may be a protein which isnormally expressed in a tumor precursor cell, but having a mutationcharacteristic of a tumor. Other antigens include mutant variant(s) ofthe normal protein having an altered activity or subcellulardistribution, e.g., mutations of genes giving rise to tumor antigens.

Specific non-limiting examples of suitable tumor antigens include: CEA,prostate specific antigen (PSA), HER-2/neu, BAGE, GAGE, MAGE 1-4, 6 and12, MUC (Mucin) (e.g., MUC-1, MUC-2, etc.), GM2 and GD2 gangliosides,ras, myc, tyrosinase, MART (melanoma antigen), Pmel 17 (gp100), GnT-Vintron V sequence (N-acetylglucoaminyltransferase V intron V sequence),Prostate Ca psm, PRAME (melanoma antigen), β-catenin, MUM-1-B (melanomaubiquitous mutated gene product), GAGE (melanoma antigen) 1, BAGE(melanoma antigen) 2-10, c-ERB2 (Her2/neu), EBNA (Epstein-Barr Virusnuclear antigen) 1-6, gp75, human papilloma virus (HPV) E6 and E7, p53,lung resistance protein (LRP), Bcl-2, and Ki-67.

Suitable cancer-associated antigens include, e.g., Melan-A/MART-1,Dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein(ADAbp), cyclophilin b, Colorectal associated antigen(CRC)-C017-1A/GA733, Carcinoembryonic Antigen (CEA) and its immunogenicepitopes CAP-1 and CAP-2, etv6, aml1, Prostate Specific Antigen (PSA)and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specificmembrane antigen (PSMA), T-cell receptor/CD3-zeta chain, MAGE-family oftumor antigens (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5,MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12,MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1,MAGE-C2, MAGE-C3, MAGE-C4, MAGE-05), GAGE-family of tumor antigens(e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8,GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53,MUC family, HER2/neu, p21ras, RCAS1, α-fetoprotein, E-cadherin,α-catenin, β-catenin and γ-catenin, p120ctn, gp100.sup.Pme1117, PRAME,NY-ESO-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1, CT-7, cdc27, adenomatous polyposis coliprotein (APC), fodrin, P1A, Connexin 37, Ig-idiotype, p15, gp75, GM2 andGD2 gangliosides, viral products such as human papilloma virus proteins,Smad family of tumor antigens, 1mp-1, EBV-encoded nuclear antigen(EBNA)-1, or c-erbB-2.

Autoantigens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, an autoantigen. In some cases, animmunomodulatory composition of the present disclosure comprises, inaddition to HKCC, one or more autoantigens, e.g., 1, 2, 3, 4, 5, or moreantigens, from one or more self tissues.

For example, where the autoimmune disease is type 1 diabetes, an antigencan be pancreatic islet beta cell associated antigen, HSP60; forsystemic lupus erythematosus, an antigen can be snRNP; for Grave'sdisease, an antigen can be thyroglobulin, thyrotropin receptor or athyroid epithelial cell; for thrombocytopenic purpura, an antigen can bea platelet, GPIIB/IIIa; for multiple sclerosis, an antigen can be myelinbasic protein, MOG, PLP; for celiac disease, an antigen can betransglutaminidase.

A suitable autoantigen can be an autoantigen involved in the initiationand/or propagation of an autoimmune disease, the pathology of which canbe due to the presence of antibodies specific for a molecule expressedby the relevant target organ, tissue, or cells, e.g., systemic lupuserythematosus (SLE) or myasthenia gravis (MG). In such diseases, it canbe desirable to direct an ongoing antibody-mediated (i.e., a Th2-type)immune response to the relevant autoantigen towards a cellular (i.e., aTh1-type) immune response. Alternatively, it can be desirable to preventonset of or decrease the level of a Th2 response to the autoantigen in asubject not having, but who is suspected of being susceptible to, therelevant autoimmune disease by prophylactically inducing a Th1 responseto the appropriate autoantigen. Autoantigens that can be included in asubject immunomodulatory composition include, without limitation: (a)with respect to SLE, the Smith protein, RNP ribonucleoprotein, and theSS-A and SS-B proteins; and (b) with respect to MG, the acetylcholinereceptor. Examples of other antigens involved in one or more types ofautoimmune response include, e.g., endogenous hormones such asluteinizing hormone, follicular stimulating hormone, testosterone,growth hormone, prolactin, and other hormones.

Other examples of suitable autoantigens include antigens associated withneurological diseases such as schizophrenia, Alzheimer's disease,depression, hypopituitarism, and cardiovascular diseases such asatherosclerosis (e.g., an antigen for atherosclerosis can be cholesterylester transfer protein, oxidized LDL, apoB210, apoB100) etc.

Allergens

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, an allergen. Suitable allergens can beobtained and/or produced using known methods. Classes of suitableallergens include, but are not limited to, pollens, animal dander otherthan cat dander, grasses, molds, dusts, antibiotics, stinging insectvenoms, and a variety of environmental (including chemicals and metals),drug and food allergens. Common tree allergens include pollens fromcottonwood, popular, ash, birch, maple, oak, elm, hickory, and pecantrees; common plant allergens include those from mugwort, ragweed,English plantain, sorrel-dock and pigweed; plant contact allergensinclude those from poison oak, poison ivy and nettles; common grassallergens include rye grass, Timothy, Johnson, Bermuda, fescue andbluegrass allergens; common allergens can also be obtained from molds orfungi such as Alternaria, Fusarium, Hormodendrum, Aspergillus,Micropolyspora, Mucor and thermophilic actinomycetes; epidermalallergens can be obtained from house or organic dusts (typically fungalin origin), from arthropods such as house mites (Dermatophagoidespteronyssinus), or from animal sources such as feathers, and dog dander;common food allergens include milk and cheese (diary), egg, wheat, nut(e.g., peanut), seafood (e.g., shellfish), pea, bean and glutenallergens; common environmental allergens include metals (nickel andgold), chemicals (formaldehyde, trinitrophenol and turpentine), Latex,rubber, fiber (cotton or wool), burlap, hair dye, cosmetic, detergentand perfume allergens; common drug allergens include local anestheticand salicylate allergens; antibiotic allergens include penicillin,tetracycline and sulfonamide allergens; and common insect allergensinclude bee, wasp and ant venom, and cockroach calyx allergens.Particularly well characterized allergens include, but are not limitedto, the major and cryptic epitopes of the Der p I allergen (Hoyne et al.(1994) Immunology 83190-195), bee venom phospholipase A2 (PLA) (Akdis etal. (1996) J. Clin. Invest. 98:1676-1683), birch pollen allergen Bet v 1(Bauer et al. (1997) Clin. Exp. Immunol. 107:536-541), and themulti-epitopic recombinant grass allergen rKBG8.3 (Cao et al. (1997)Immunology 90:46-51). These and other suitable allergens arecommercially available and/or can be readily prepared as extractsfollowing known techniques.

Suitable allergens include tree pollen allergens, weed pollen allergens,herb pollen allergens, grass pollen allergens, mile allergens, insectallergens, venom allergens, animal hair allergens, dander allergens andfood allergens.

In some cases, the allergen is in the form of an extract, a purifiedallergen, a modified allergen or a recombinant allergen or a mutant of arecombinant allergen or any combination thereof. In some cases, theallergen is selected from the group consisting of grass pollen allergen,dust mite allergen, ragweed allergen, cat allergen and birch allergen.

An allergen can be present in an immunomodulatory composition of thepresent disclosure in an amount of from about 2.5 μg to about 75 μg perunit dosage form. For example, an allergen can be present in animmunomodulatory composition of the present disclosure in an amount offrom about 2.5 μg to about 5 μg, from about 5 μg to about 10 μg, fromabout 10 μg to about 15 μg, from about 15 μg to about 20 μg, from about20 μg to about 25 μg, from about 25 μg to about 50 μg, or from about 50μg to about 75 μg, or more than 75 μg, per unit dosage form.

In some cases, a dose of an immunomodulatory composition of the presentdisclosure that comprises an allergen has a potency of about 65 to about17,600 Biological Allergen Units (BAU). In some cases, a dose of animmunomodulatory composition of the present disclosure that comprises anallergen comprises from about 650 BAU to about 6,000 BAU.

Antibodies

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, an antibody against a cancer antigen ora pathogenic antigen (e.g., a therapeutic antibody, monoclonalantibodies, bispecific antibodies, chemoimmuno conjugated antibodies,radioimmunoconjugated antibodies, antibody-cytokine fusion proteins,antibody-antigen fusion proteins, antibody-immunotoxin fusion proteinetc.).

Antibodies that can be included in an immunomodulatory composition ofthe present disclosure include, without limitation, antibodies directedagainst co-stimulatory or co-inhibitory molecules (CD28, CD40, CTLA-4,PD-1 etc.); and other therapeutic antibodies.

Non-limiting examples of suitable antibodies include, but are notlimited to, adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab,bapineuzumab, basiliximab, belimumab, briakinumab, brodalumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, etrolizumab, gemtuzumab ozogamicin, golimumab, ibritumomabtiuxetan, labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumornab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

Non-limiting examples of therapeutic and prophylactic antibodies thatcan be used in combination with an immunomodulatory composition of thepresent disclosure include MDX-010 (Medarex, N.J.) which is a humanizedanti-CTLA-4 antibody for the treatment of prostate cancer; SYNAGIS™(MedImmune, Md.) which is a humanized anti-respiratory syncytial virus(RSV) monoclonal antibody for the treatment of RSV infection; andHERCEPTIN™ (Trastuzumab) (Genentech, Calif.) which is a humanizedanti-HER2 monoclonal antibody for the treatment of metastatic breastcancer. Other examples are humanized anti-CD18 F(ab′)₂ (Genentech);CDP860 which is a humanized anti-CD18 F(ab′)₂ (Celltech, UK); PRO542which is an anti-HIV gp120 antibody fused with CD4 (Progenics/GenzymeTransgenics); Ostavir which is a human anti-Hepatitis B virus antibody(Protein Design Lab/Novartis); PROTOVIR™ which is a humanized anti-CMVIgGI antibody (Protein Design Lab/Novartis); MAK-195 (SEGARD) which is amurine anti-TNF-α F(ab′)₂ (Knoll Pharma/BASF); IC14 which is ananti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody(Genentech); OVAREX™ which is a murine anti-CA 125 antibody (Altarex);PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2aantibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype(GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is achimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which is ahumanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti-CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics);Smart ID10 which is a humanized anti-HLA antibody (Protein Design Lab);ONCOLYM™ (Lym-1) is a radiolabelled murine anti-HLA DIAGNOSTIC REAGENTantibody (Techniclone); ABX-IL8 is a human anti-IL8 antibody (Abgenix);anti-CD11a is a humanized IgG1 antibody (Genentech/Xoma); ICM3 is ahumanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatizedanti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelledmurine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanizedanti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A isa humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is ahumanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanizedanti-VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64 (FcγR)antibody (Medarex/Centeon); SCH55700 is a humanized anti-IL-5 IgG4antibody (Celltech/Schering); SB-240563 and SB-240683 are humanizedanti-IL-5 and IL-4 antibodies, respectively, (SmithKline Beecham);rhuMab-E25 is a humanized anti-IgE IgG1 antibody(Genentech/Norvartis/Tanox Biosystems); ABX-CBL is a murine anti CD-147IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(MedImmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′).sub.2(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor). The above-listed immunoreactive reagents, as wellas any other immunoreactive reagents, may be administered according toany regimen known to those of skill in the art, including the regimensrecommended by the suppliers of the immunoreactive reagents.

Cytokines

In some cases, an immunomodulatory composition of the present disclosurecomprises, in addition to HKCC, a cytokine. Cytokines that can beincluded in an immunomodulatory composition of the present disclosureinclude, without limitation, interleukins, transforming growth factors(TGFs), fibroblast growth factors (FGFs), platelet derived growthfactors (PDGFs), epidermal growth factors (EGFs), colony stimulatingfactors (CSFs), connective tissue activated peptides (CTAPs), osteogenicfactors, and biologically active analogs, fragments, and derivatives ofsuch growth factors. Suitable cytokines include B/T-cell differentiationfactors, B/T-cell growth factors, mitogenic cytokines, chemotacticcytokines, colony stimulating factors, angiogenesis factors, IFN-α,IFN-β, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11,IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL22, etc., leptin, myostatin,macrophage stimulating protein, platelet-derived growth factor, tumornecrosis factor (TNF)-alpha (TNF-α), TNF-β, nerve growth factor (NGF),CD40L, CD137L/4-1BBL, human lymphotoxin-β, G-CSF, M-CSF, GM-CSF,platelet-derived growth factor (PDGF), IL-1α, IL-1β, IP-10, PF4, GRO,9E3, erythropoietin, endostatin, angiostatin, vascular endothelialgrowth factor (VEGF) or any fragments or combinations thereof. Othercytokines include members of the transforming growth factor (TGF)supergene family include the beta transforming growth factors (forexample TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (forexample, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);heparin-binding growth factors (for example, fibroblast growth factor(FGF), epidermal growth factor (EGF), platelet-derived growth factor(PDGF), insulin-like growth factor (IGF)); hematopoietic growth factors(Flt3); pituitary growth hormones or derivatives; growth hormones,neuroactive hormones, Inhibins (for example, Inhibin A, Inhibin B);differentiation factors (for example, GDF-1); and Activins (for example,Activin A, Activin B, Activin AB). In some cases, an immunomodulatorycomposition of the present disclosure comprises, in addition to HKCC, acompound or agent modulating cytokines.

Caulobacter crescentus

An immunomodulatory composition of the present disclosure comprisesinactivated Caulobacter, where the Caulobacter is non-pathogenic. Thenon-pathogenic Caulobacter genus includes 19 different species,including two species of Asliccacaulis (C. vibroides, C. henricii, C.intermedius, C. robiginosus, C. rutilis, C. subvibriodes, C. fusiformis,C. rossii, A. excentricus, A. biprosthecum etc.). See, e.g., JSPoindexter, The Caulobacters: Ubiquitous Unusual Bacteria, Microbiol Rev45, 123-179, 1981). Several of the Caulobacter sp. are available fromthe American Type Culture Collection (ATCC), such as CB35, CB26, CB28,KA5, CB66, FC4 etc. All of these species of Caulobacter in heat-killed,inactivated, mutated or attenuated forms can be used as immunomodulatoryagents described herein. In addition, Caulobacter bacteria can be innon-motile prosthecate, motile swarmer, stubby flagellin and flagellinpositive, flagellin negative, dividing and/or non-dividing forms.Caulobacter sp. can be grown at temperatures ranging from 18°-42° C.,and pH ranging from 5-9, but optimally at a temperature in a range of23-25° C. and pH 7.

Mutated or genetically modified forms of Caulobacter sp. can be producedby modifying the nutrients, chemicals, pH, temperature, ultraviolet orinfrared light, radiation etc. of the culture conditions, or geneticallymodifying various enzymes, metabolic pathways, surface molecules,nucleic acids, plasmids, cellular and cell wall components, smooth andrough LPS in live bacteria (JS Poindexter, The Caulobacters: UbiquitousUnusual Bacteria, Microbiol Rev 45, 123-179, 1981).

Caulobacter crescentus can act as a carrier and/or delivery vehicle todeliver antigens. As a non genetic modification (GM), such aselectrostatic and hydrophobic interactions, binding of antigens to theCaulobacter crescentus surface may enable the Caulobacter crescentus toact as an antigen carrier and/or delivery vehicle. Further, due tobioadhesion/mucoadhesion, Caulobacter crescentus may facilitate antigenuptake by M cell transport, delivery to and subsequentactivation/maturation of DCs/APCs, induction of NK, NKT, B and T cellresponses at mucosal surfaces.

Although the discussion below focuses on Caulobacter crescentus, any ofa variety of non-pathogenic Caulobacter species can be included in animmunomodulatory composition of the present disclosure.

In some cases, an immunomodulatory composition of the present disclosurecomprises heat-killed Caulobacter crescentus (HKCC). In some cases, theCaulobacter crescentus is wild-type. In some cases, the Caulobactercrescentus is a lipopolysaccharide-negative strain. In some cases, theCaulobacter crescentus is an S-layer-negative strain. In some cases, theHKCC is mutated attenuated, or contains suicidal mutations. In somecases CC is chemically or physically inactivated. In some cases,Caulobacter crescentus is with or without a drug resistant plasmid suchas chloramphenicol, penicillin resistant plasmids.

In some cases, the Caulobacter crescentus is genetically modified toproduce one or more heterologous polypeptides. The polypeptides can beof a wide range of sizes. Suitable heterologous polypeptides include,but are not limited to, CD40, a costimulatory protein found onantigen-presenting cells or T cells; DEC205 (see, e.g. Lahoud et al.(2012) Proc. Natl. Acad. Sci. USA 109:16270); CD40L; a co-inhibitoryprotein found on antigen-presenting cells (APCs) or T cells; a cytokine(e.g., GM-CSF; or any of the above-listed cytokines); a chemokine; anantigen (e.g., a viral antigen; a bacterial antigen; a tumor-associatedantigen; a helminth antigen; a protozoan antigen; an autoantigen asdescribed herein above); an antibody against an antigen (e.g., a viralantigen; a bacterial antigen; a tumor-associated antigen; a helminthantigen; a protozoan antigen; as described herein above), a signallingmolecule, a receptor, a cytokine; a fusion protein (e.g., an antigen anda cytokine, an antigen and a carrier protein) etc. In some cases,Caulobacter crescentus is genetically modified to express anticancer(e.g., kinesin spindle protein), antiviral (e.g., entry and fusioninhibitors), antibacterial, antifungal and/or antimicrobial peptides onthe surface, in secreted form or intracellularly.

In some cases, Caulobacter crescentus is modified by labeling orcoupling the bacterium with fluorescent, radioactive isotope, light tagsetc.

In some cases, Caulobacter crescentus is genetically modified to providedesired immune responses. In some cases, Caulobacter crescentus isgenetically modified so that microbe is attenuated. In some cases, thenucleic acid of the Caulobacter crescentus is modified so that microbeis attenuated for proliferation.

In some cases, an immunomodulatory composition of the present disclosurecomprises whole HKCC. In some cases, an immunomodulatory composition ofthe present disclosure comprises individual or multiple components ofHKCC which can be isolated, synthesized, or genetically manufactured.Fractions of inactivated Caulobacter crescentus can be obtained bytreatment with various organic solvents, enzymes such as glycosidases,lipase, DNAse, RNAse, protease, lysozyme etc.

In some cases, Caulobacter crescentus is bioengineered in its outermembrane vesicle to package and deliver chemotherapeutics and/orimmunotherapeutics.

Adjuvants

An immunomodulatory composition of the present disclosure can comprise,in addition to HKCC, one or more additional adjuvants.

Exemplary additional adjuvants include, but are not limited to: (1)oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) MF59™ (WO90/14837; Chapter 10 in Vaccine design: the subunit and adjuvantapproach, eds. Powell & Newman, Plenum Press 1995), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE)formulated into submicron particles using a microfluidizer, (b) SAF,containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) RIBI™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentssuch as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cellwall skeleton (CWS), e.g., MPL+CWS (Detox™); (2) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may beused or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-28, etc.) (WO99/44636), etc.),interferons (e.g. gamma interferon), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), colony-stimulating factors(e.g., GM-CSF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylatedMPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19,618-622; WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO98/55495, WO 98/37919 and WO 98/52581), i.e., oligonucleotidescontaining at least one CG dinucleotide, where the cytosine isunmethylated; (8) a polyoxyethylene ether or a polyoxyethylene estere.g. WO 99/52549; (9) a polyoxyethylene sorbitan ester surfactant incombination with an octoxynol (WO 01/21207) or a polyoxyethylene alkylether or ester surfactant in combination with at least one additionalnon-ionic surfactant such as an octoxynol (WO 01/21152); (10) a saponinand an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)(WO 00/62800); (11) an immunostimulant and a particle of metal salt e.g.WO 00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally including asterol) e.g. WO 98/57659; (14) alphaGalCer and its derivatives; (16)toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists,RIG-I agonists, agonists for C-type lectin receptors and other pathogenrecognition receptor (PRR) agonists e.g., CpG ODNs, ISS-ODNs,rinatolimod, polyI:C and its derivatives, flagellin, ampligen,imidazoquinalines (e.g., imiquimod, resiquimod), muramyl dipeptides;(17) other substances that act as immunostimulating agents to enhancethe efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Adjuvants suitable for administration to a human includedin some cases.

Further exemplary additional adjuvants include, but are not limited to:cholera toxin B subunit, BCG, Pseudomonas aeruginosa exoprotein A,tocopherol, HBV core, E. coli heat labile toxins (such as LT-A, LT-B),Pertussis toxin, Diphtheria toxoid, tetanus toxoid, Cholera toxinderived (CTA1-DD, CT), mutant LT and CT, Aluminium salt-based adjuvants(such as Alum, Aluminum phosphate, Aluminum sulphate, Alhydrogel),Calcium phosphate, kaolin, monophosphoryl lipid Λ (MPL^(R)) and itsderivatives, glucoppyranosyl lipid Λ, synthetic lipid Λ, Lipid Amimetics, Vitamin E, Depovax™, Saponins (Quil-A, AS01, AS02(squalene+MPL+QS-21)), ΛS03, ΛS04 (alum+MPL^(R)), Tomatin, Protolin,RC-529, Pluronic™, Monatides, Matrix-M, OM-174, Lipovac, IC-31,bacterial/mycobacterial peptides (such as KLK, cationic (poly)peptides,anti-bacterial microbial peptides, defensins, tuftsin, cathelicidin),dipeptides (such as pidotimod), Bestatin, Hepon (tetradecapeptide),SCV-07 (gamma-D-glutamyl-L-tryptophan), Thymosin-a, Immunofan, Thymogen,Indolicidin and its derivatives, polyphosphagene and its derivatives,Gellan, nucleotides (mononucleotides, dinucleotides, polynucleotides,cyclic nucleotides), Eurocine etc.

An immunomodulatory composition of the present disclosure can comprise,in addition to HKCC, one or more mucoadhesives such as sodium alginate,starch, lectins, thiolated polymers, GelVac™, sodiumcarboxymethylcellulose, hydroxylpropyl methylcellulose, carbomers, cetyltrimethyl ammonium bromide.

An immunomodulatory composition of the present disclosure can comprise,in addition to HKCC, one or more additional adjuvant formulations suchas oil-in-water emulsions, water-in-oil emulsions, nanoemulsions,particulate delivery systems, liposomes, microspheres, biodegradablemicrospheres, patches virosomes, proteoliposomes, proteasomes,Immunostimulatory complexes (ISCOMs, ISCOMATRIX), microparticles,nanoparticles, biodegradable nanoparticles, silicon nanoparticles,polymeric micro/nano particles, polymeric lamellar substrate particles(PLSP), microparticle resins, nanolipogels, synthetic/biodegradable andbiocompatible semisynthetic or natural polymers or dendrimers (such asPLG, PLGA, PLA, polycaprolactone, silicone polymer, polyesters,poly-dimethyl siloxane, sodium polystyrene sulphonate, polystyrenebenzyl trimethyl ammonium chloride, polystyrene divinyl benzene resin,polyphosphazene, poly-[di-(carboxylactophenoxy)phosphazene] (PCPP),poly-(methylmethacrylate), dextran, polyvinylpyrrolidone, hyaluronicacid and derivatives, chitosan and its derivatives, polysaccharides,Delta inulin polysaccharide, glycolipids (synthetic or natural),lipopolysaccharides, polycationic compound(s) (such as Poly-amino acids,poly-(γ-glutamic acid), poly-arginine-HCl, poly-L-lysine, polypeptides,biopolymers), cationic dimethyldioctadecyl ammonium (DDA),alpha-galactosyl ceramide and its derivatives, archaeal lipids andderivatives, lactanes, gallen, glycerolipids, phospholipids, cochleates,etc. or mixtures thereof.

An immunomodulatory composition of the present disclosure can comprise,in addition to HKCC, one or more additional adjuvant formulations suchas oil-in-water emulsions or water-in-oil emulsions including edibleoils (such as olive oil, mustard oil, vegetable oil, soybean oil,mineral oil etc.).

An immunomodulatory composition of the present disclosure can comprise,in addition to HKCC, one or more additional surfactants and detergents(e.g., non-ionic detergents or niosomes) (such as Tween-80, Polysorbate80, Span 85, Stearyl tyrosine etc.). An immunomodulatory composition ofthe present disclosure can comprise, in addition to HKCC, an additionalcomponent or adjuvant mentioned above which provides a depot effect.

Methods

The present disclosure provides methods of modulating an immune responsein an individual, the method comprising administering to the individualan effective amount of an immunomodulatory composition of the presentdisclosure.

The present disclosure also provides a method of enhancing antigenpresentation on a dendritic cell, the method comprising: a) contactingdendritic cells (DCs) obtained from an individual with a compositioncomprising: i) heat-killed Caulobacter crescentus; and ii) an antigen;the contacting step is in vitro, and enhances antigen presentation ofthe antigen on the DCs, thereby generating a population ofantigen-presenting DCs. The population of antigen-presenting DCs canthen be administered to the individual from whom the DCs were obtained.

In some cases, various immune cells can be obtained from lymphoidtissues, peripheral blood, organs and tissues, and/or can bedifferentiated from stem cells obtained from bone marrow or variousorgans.

The present disclosure also provides a method of inducing proliferationand/or differentiation of stem cells, the method comprising contactingstem cells obtained from an individual with a composition comprisingheat-killed Caulobacter crescentus. Contacting the stem cells with theHKCC leads to proliferation and differentiation of the stem cells,thereby generating a population of expanded and differentiated cells.The population of expanded and differentiated cells can then beadministered to the individual from whom the stem cells were obtained.

The present disclosure further provides a method of activating effectorlymphocytes such as NK, NKT, T cells, and B cells, the methodcomprising: a) contacting effector cells (NK, NKT, T cells, B cells)obtained from an individual with a composition comprising: i)heat-killed Caulobacter crescentus; and/or ii) an antigen in thepresence or absence of antigen presenting cells. Contacting the effectorlymphocytes with the HKCC enhances activation of the effectorlymphocytes, thereby generating a population of activated effectorlymphocytes. The population of activated effector lymphocytes can thenbe administered to the individual from whom the lymphocytes wereobtained.

Methods of Modulating an Immune Response

The present disclosure provides methods of modulating an immune responsein an individual, the method comprising administering to the individualan effective amount of an immunomodulatory composition of the presentdisclosure.

In some cases, the immune response is a humoral immune response. In somecases, the present disclosure provides methods of enhancing a humoralimmune response in an individual, the method comprising administering tothe individual an effective amount of an immunomodulatory composition ofthe present disclosure. In some cases, the immunomodulatory compositiondoes not include any additional antigens (other than antigens present onHKCC). In some cases, the immunomodulatory composition comprises anantigen (e.g., an antigen other than antigens present on HKCC). Asdescribed above, suitable antigens include bacterial antigens, viralantigens, tumor-associated antigens, protozoan antigens, and helminthantigens.

In some cases, the immune response is a cellular immune response. Insome cases, the present disclosure provides methods of enhancing acellular immune response in an individual, the method comprisingadministering to the individual an effective amount of animmunomodulatory composition of the present disclosure. In some cases,the immunomodulatory composition does not include any additionalantigens (other than antigens present on HKCC). In some cases, theimmunomodulatory composition comprises an antigen (e.g., an antigenother than antigens present on HKCC). As described above, suitableantigens include bacterial antigens, viral antigens, tumor-associatedantigens, protozoan antigens, and helminth antigens.

In some cases, the immune response comprises an increase in the numberof B cells. In some cases, a subject method comprising administering toan individual in need thereof an effective amount of an immunomodulatorycomposition, where an effective amount of an immunomodulatorycomposition is an amount that, when administered to the individual in asingle dose or in multiple doses, is effective to increase the number ofB cells in an individual. For example, in some cases, an effectiveamount of an immunomodulatory composition of the present disclosure isan amount that is effective, when administered in a single dose or inmultiple doses, to increase the number of B cells in an individual by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least10-fold, or more than 10-fold, compared to the number of B cells in theindividual in the absence of treatment with the immunomodulatorycomposition. In some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase thenumber of antigen-specific B cells in an individual. For example, insome cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number ofantigen-specific B cells in an individual by at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 10-fold, or more than 10-fold,compared to the number of antigen-specific B cells in the individual inthe absence of treatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase activation of B cellsin an individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase activation of B cells in an individual by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, at least25-fold, at least 50-fold, at least 100-fold, or more than 100-fold,compared to the activation level of B cells in the individual in theabsence of treatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the amount ofantibody specific to a given antigen in the individual. For example, insome cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the amount ofantibody specific to a given antigen in an individual by at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, or morethan 10-fold, compared to the amount of antibody specific to the antigenin the individual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase production of one ormore cytokines in the individual. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase production of one or morecytokines in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, or more than 10-fold, compared to theamount of the cytokine in the individual in the absence of treatmentwith the immunomodulatory composition. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase production of GM-CSF in anindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, or more than 10-fold, compared to the amountof GM-CSF in the individual in the absence of treatment with theimmunomodulatory composition. For example, in some cases, an effectiveamount of an immunomodulatory composition of the present disclosure isan amount that is effective, when administered in a single dose or inmultiple doses, to increase production of IL-22 in an individual by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least10-fold, or more than 10-fold, compared to the amount of IL-22 in theindividual in the absence of treatment with the immunomodulatorycomposition. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase production of interferon (IFN)-α and/or IFN-β and/or IFN-γin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, or more than 10-fold, compared to theamount of IFN-α or IFN-β or IFN-γ in the individual in the absence oftreatment with the immunomodulatory composition. For example, in somecases, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase production of one ormore of IL-17A, IL-2, IL-10, IL-6 and/or TNF-α in an individual by atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least10-fold, or more than 10-fold, compared to the amount of IL-17A, IL-2,IL-10, IL-6, or TNF-α in the individual in the absence of treatment withthe immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase a Th1 response in anindividual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase a Th1 response in an individual by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, more than10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 100-fold, or more than 100-fold, compared to the levelof the Th1 response in the individual in the absence of treatment withthe immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of CD4⁺ T cells in an individual. For example, in some cases,an effective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofCD4⁺ T cells in an individual by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least100-fold, or more than 100-fold, compared to the number and/or activityof CD4⁺ T cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD4⁺ T cellsin an individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD4⁺ T cellsin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the number and/or activity ofantigen-specific CD4⁺ T cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of CD8⁺ T cells in an individual. For example, in some cases,an effective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofCD8⁺ T cells in an individual by at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 75%, at least 100% (or 2-fold), at least2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, at least15-fold, at least 20-fold, at least 25-fold, at least 50-fold, at least100-fold, or more than 100-fold, compared to the number and/or activityof CD8⁺ T cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD8⁺ T cellsin an individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific CD8⁺ T cellsin an individual by at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the number and/or activity ofantigen-specific CD8⁺ T cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of cytolytic T cells in an individual. For example, in somecases, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase the number and/oractivity of cytolytic T cells in an individual by at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, more than10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 100-fold, or more than 100-fold, compared to thenumber and/or activity of cytolytic T cells in the individual in theabsence of treatment with the immunomodulatory composition. In somecases, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase the number and/oractivity of antigen-specific cytolytic T cells in an individual. Forexample, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase thenumber and/or activity of antigen-specific cytolytic T cells in anindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity ofantigen-specific cytolytic T cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of one or more of natural killer (NK) cells, NKT cells,macrophages, and dendritic cells (DCs) in an individual. For example, insome cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of one or more of NK cells, NKT cells, macrophages, and DCs inan individual by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity of one or more ofNK cells, NKT cells, macrophages, and DCs in the individual in theabsence of treatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase, decrease or balancethe number and/or function of Tregs in an individual. Tregs (regulatoryT cells) are CD4⁺ or CD8⁺, and may also be FoxP3⁺. T_(regs) may also bedefined by other markers such as PD-1, CTLA-4 etc. Regulatory cells mayalso be comprised of other innate cells such as NK, NKT and DCs, and Blymphocytes. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to modulate the number of Tregs in an individual by at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, or more than 75%,compared to the number of Tregs in the individual in the absence oftreatment with the immunomodulatory composition. For example, in somecases, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to reduce the number of Tregs in anindividual by at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, or more than 75%, compared to the number of Tregs in theindividual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of Th17 cells in an individual. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofTh17 cells in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, more than 10-fold, at least 15-fold,at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold,or more than 100-fold, compared to the number and/or activity of Th17cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th17 cells inan individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th17 cells inan individual by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity ofantigen-specific Th17 cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to increase the number and/oractivity of Th22 cells in an individual. For example, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase the number and/or activity ofTh22 cells in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, more than 10-fold, at least 15-fold,at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold,or more than 100-fold, compared to the number and/or activity of Th22cells in the individual in the absence of treatment with theimmunomodulatory composition. In some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th22 cells inan individual. For example, in some cases, an effective amount of animmunomodulatory composition of the present disclosure is an amount thatis effective, when administered in a single dose or in multiple doses,to increase the number and/or activity of antigen-specific Th22 cells inan individual by at least 10%, at least 15%, at least 20%, at least 25%,at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the number and/or activity ofantigen-specific Th22 cells in the individual in the absence oftreatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to elicit, boost and/or regulateinnate and/or adaptive (including both cellular and humoral) immuneresponses in an individual. For example, in some cases, an effectiveamount of an immunomodulatory composition of the present disclosure isan amount that is effective, when administered in a single dose or inmultiple doses, to modulate the number and/or activity of innate and/oradaptive immune cells and/or their effector functions in an individualby at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,at least 35%, at least 40%, at least 45%, at least 50%, at least 75%, atleast 100% (or 2-fold), at least 2.5-fold, at least 5-fold, at least10-fold, more than 10-fold, at least 15-fold, at least 20-fold, at least25-fold, at least 50-fold, at least 100-fold, or more than 100-fold,compared to the number and/or activity of one or more of innate oradaptive immune cells and/or their effector functions in the individualin the absence of treatment with the immunomodulatory composition.

In some cases, an effective amount of an immunomodulatory composition ofthe present disclosure is an amount that is effective, when administeredin a single dose or in multiple doses, to protect innate and/or adaptiveimmune cells from depletion or prevent their apoptosis in an individual.For example, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to protectinnate and/or adaptive immune cells from depletion or prevent theirapoptosis in an individual by at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold,at least 5-fold, at least 10-fold, more than 10-fold, at least 15-fold,at least 20-fold, at least 25-fold, at least 50-fold, at least 100-fold,or more than 100-fold, compared to the number and/or activity of one ormore of innate or adaptive immune cells and/or their effector functionsin the individual in the absence of treatment with the immunomodulatorycomposition.

In some cases, an immunomodulatory composition of the present disclosurecomprises HKCC and an antigen. Where an immunomodulatory composition ofthe present disclosure comprises HKCC and an antigen, in some cases, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase an immune response to the antigenby at least about 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the immune response to the antigen in theabsence of treatment with the immunomodulatory composition. For example,where the antigen is an antigen associated with or derived from a cancercell, a pathogenic bacterium, a pathogenic virus, or a pathogenicprotozoan, an effective amount of an immunomodulatory composition of thepresent disclosure is an amount that is effective, when administered ina single dose or in multiple doses, to increase an immune response tothe antigen by at least about 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 75%, at least 100% (or 2-fold), at least 2.5-fold, atleast 5-fold, at least 10-fold, more than 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 50-fold, at least 100-fold, ormore than 100-fold, compared to the immune response to the antigen inthe absence of treatment with the immunomodulatory composition. Theimmune response can be a humoral immune response, e.g., a B cell orantibody immune response. Thus, e.g., in some cases, where the antigenis an antigen associated with or derived from a cancer cell, apathogenic bacterium, a pathogenic virus, or a pathogenic protozoan, aneffective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to increase a B cell response to the antigenby at least about 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 75%, at least 100% (or 2-fold), at least 2.5-fold, at least5-fold, at least 10-fold, more than 10-fold, at least 15-fold, at least20-fold, at least 25-fold, at least 50-fold, at least 100-fold, or morethan 100-fold, compared to the B cell response to the antigen in theabsence of treatment with the immunomodulatory composition. For example,in some cases, where the antigen is an antigen associated with orderived from a cancer cell, a pathogenic bacterium, a pathogenic virus,or a pathogenic protozoan, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase theamount of antibody specific to the antigen by at least about 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 75%, at least 100% (or2-fold), at least 2.5-fold, at least 5-fold, at least 10-fold, more than10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least50-fold, at least 100-fold, or more than 100-fold, compared to theamount of antibody specific to the antigen in the absence of treatmentwith the immunomodulatory composition. The immune response can be acellular immune response, e.g., a T cell immune response. Thus, e.g., insome cases, where the antigen is an antigen associated with or derivedfrom a cancer, a pathogenic bacterium, a pathogenic virus, or apathogenic protozoan, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to increase a Tcell response to the antigen by at least about 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, atleast 100-fold, or more than 100-fold, compared to the T cell responseto the antigen in the absence of treatment with the immunomodulatorycomposition. In some cases, the immune response is a humoral immuneresponse and a cellular immune response.

Adjuvants

In some embodiments, a subject method involves administration of asubject immunomodulatory composition, where the immunomodulatorycomposition comprises HKCC and one or more additional adjuvants.

Exemplary additional adjuvants include, but are not limited to: (1)oil-in-water emulsion formulations (with or without other specificimmunostimulating agents such as muramyl peptides (see below) orbacterial cell wall components), such as for example (a) MF59™ (WO90/14837; Chapter 10 in Vaccine design: the subunit and adjuvantapproach, eds. Powell & Newman, Plenum Press 1995), containing 5%Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing MTP-PE)formulated into submicron particles using a microfluidizer, (b) SAF,containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymerL121, and thr-MDP either microfluidized into a submicron emulsion orvortexed to generate a larger particle size emulsion, and (c) RIBI™adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentssuch as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cellwall skeleton (CWS), e.g., MPL+CWS (Detox™); (2) saponin adjuvants, suchas QS21 or Stimulon™ (Cambridge Bioscience, Worcester, Mass.) may beused or particles generated therefrom such as ISCOMs (immunostimulatingcomplexes), which ISCOMS may be devoid of additional detergent e.g. WO00/07621; (3) Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA); (4) cytokines, such as interleukins (e.g. IL-1, IL-2,IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-28, etc.) (WO99/44636), etc.),interferons (e.g. gamma interferon), macrophage colony stimulatingfactor (M-CSF), tumor necrosis factor (TNF), colony-stimulating factors(e.g., GM-CSF), etc.; (5) monophosphoryl lipid A (MPL) or 3-O-deacylatedMPL (3dMPL) e.g. GB-2220221, EP-A-0689454, optionally in the substantialabsence of alum when used with pneumococcal saccharides e.g. WO00/56358; (6) combinations of 3dMPL with, for example, QS21 and/oroil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231;(7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19,618-622; WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO98/55495, WO 98/37919 and WO 98/52581), i.e., oligonucleotidescontaining at least one CG dinucleotide, where the cytosine isunmethylated; (8) a polyoxyethylene ether or a polyoxyethylene estere.g. WO 99/52549; (9) a polyoxyethylene sorbitan ester surfactant incombination with an octoxynol (WO 01/21207) or a polyoxyethylene alkylether or ester surfactant in combination with at least one additionalnon-ionic surfactant such as an octoxynol (WO 01/21152); (10) a saponinand an immunostimulatory oligonucleotide (e.g. a CpG oligonucleotide)(WO 00/62800); (11) an immunostimulant and a particle of metal salt e.g.WO 00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO99/11241; (13) a saponin (e.g. QS21)+3dMPL+IM2 (optionally including asterol) e.g. WO 98/57659; (14) alphaGalCer and its derivatives; (16)toll-like receptor (TLR) agonists, NOD-like receptor (NLR) agonists,RIG-I agonists, agonists for C-type lectin receptors and other pathogenrecognition receptor (PRR) agonists e.g., CpG ODNs, ISS-ODNs,rinatolimod, polyI:C and its derivatives, flagellin, ampligen,imidazoquinalines (e.g., imiquimod, resiquimod), muramyl dipeptides;(17) other substances that act as immunostimulating agents to enhancethe efficacy of the composition. Muramyl peptides includeN-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamineMTP-PE), etc. Adjuvants suitable for administration to a human includedin some cases.

Further exemplary additional adjuvants include, but are not limited to:cholera toxin B subunit, BCG, Pseudomonas aeruginosa exoprotein A,tocopherol, HBV core, E. coli heat labile toxins (such as LT-A, LT-B),Pertussis toxin, Diphtheria toxoid, tetanus toxoid, Cholera toxinderived (CTA1-DD, CT), mutant LT and CT, Aluminium salt-based adjuvants(such as Alum, Aluminum phosphate, Aluminum sulphate, Alhydrogel),Calcium phosphate, kaolin, monophosphoryl lipid A (MPL^(R)) and itsderivatives, glucoppyranosyl lipid A, synthetic lipid A, Lipid Amimetics, Vitamin E, Depovax™, Saponins (Quil-A, AS01, AS02(squalene+MPL+QS-21)), AS03, AS04 (alum+MPL^(R)), Tomatin, Protolin,RC-529, Pluronic™, Monatides, Matrix-M, OM-174, Lipovac, IC-31,bacterial/mycobacterial peptides (such as KLK, cationic (poly)peptides,anti-bacterial microbial peptides, defensins, tuftsin, cathelicidin),dipeptides (such as pidotimod), Bestatin, Hepon (tetradecapeptide),SCV-07 (gamma-D-glutamyl-L-tryptophan), Thymosin-a, Immunofan, Thymogen,Indolicidin and its derivatives, polyphosphagene and its derivatives,Gellan, nucleotides (mononucleotides, dinucleotides, polynucleotides,cyclic nucleotides), Eurocine etc.

Combination Therapy

In some embodiments, a subject method involves administration of asubject immunomodulatory composition as monotherapy, e.g.,administration of a subject immunomodulatory composition only, withoutco-administration of any other therapeutic agent. In other embodiments,a subject treatment method is a combination therapy involvingadministration of: a) a subject immunomodulatory composition; and b) atleast one additional therapeutic agent (or a pharmaceutically acceptablesalt, prodrugs, salts of prodrugs, stereoisomers, tautomers etc. of thetherapeutic agent), where the immunomodulatory composition and the atleast one additional therapeutic agent are administered in combinedamounts that are effective to modulate an immune response. Suitableadditional therapeutic agents are described below.

A subject combination therapy can involve: a) administration of animmunomodulatory composition and at least one additional therapeuticagent at the same time, in the same formulation or in separateformulations; b) administration of at least one additional therapeuticagent within about 5 minutes to about 4 weeks of administration of animmunomodulatory composition, e.g., administration of at least oneadditional therapeutic agent within about 5 minutes to about 15 minutes,within about 15 minutes to about 30 minutes, within about 30 minutes toabout 60 minutes, within about 1 hour to about 2 hours, within about 2hours to about 4 hours, within about 4 hours to about 8 hours, withinabout 8 hours to about 12 hours, within about 12 hours to about 24hours, within about 24 hours to about 2 days, within about 2 days toabout 4 days, within about 4 days to about 7 days, within about 1 weekto about 2 weeks, or within about 2 weeks to about 4 weeks ofadministration of an immunomodulatory composition.

In some embodiments, the at least one additional therapeutic agent isco-formulated with the immunomodulatory composition. In otherembodiments, the at least one additional therapeutic agent and theimmunomodulatory composition are separately formulated.

In some embodiments, an effective amount of an immunomodulatorycomposition and an at least one additional therapeutic agent aresynergistic amounts. As used herein, a “synergistic combination” or a“synergistic amount” of a subject immunomodulatory composition and anadditional (e.g., a second) therapeutic agent is a combination or amountthat is more effective in the therapeutic or prophylactic treatment of adisease than the incremental improvement in treatment outcome that couldbe predicted or expected from a merely additive combination of (i) thetherapeutic or prophylactic benefit of the immunomodulatory compositionwhen administered at that same dosage as a monotherapy and (ii) thetherapeutic or prophylactic benefit of the additional therapeutic agentwhen administered at the same dosage as a monotherapy.

A subject combination therapy can involve: administration of animmunomodulatory composition and at least one additional form of therapysuch as radiation therapy (comprising radioisotopes such as ¹²⁵I,strontium-89, ³²P, alpha-emitting isotopes, beta-emitting isotopesetc.), photodynamic therapy, laser therapy, natural product therapy,nutraceutical therapy, cellular therapy, prebiotic therapy, probiotictherapy, symbiotic therapy, paraprobiotic therapy etc., given at thesame or different times.

In some embodiments, an effective amount of an immunomodulatorycomposition can be administered in a heterologous or homologousprime-boost vaccine, immunotherapy and/or chemotherapy regimen(s).

A subject combination therapy can involve: administration of animmunomodulatory composition and a therapeutic vaccine.

A subject combination therapy can involve: administration of animmunomodulatory composition and a therapeutic antibody. For example, insome embodiments, a subject method involves: a) administration of animmunomodulatory composition of the present disclosure; and b)administration of at least one antibody. The HKCC and the antibody canbe in the same formulation or in separate formulations. The HKCC and theantibody can be administered simultaneously, or at different times.Suitable antibodies include an antibody against a cancer antigen or apathogenic antigen (e.g., a therapeutic antibody, monoclonal antibodies,bispecific antibodies, chemoimmuno conjugated antibodies,radioimmunoconjugated antibodies, antibody-cytokine fusion proteins,antibody-antigen fusion proteins, antibody-immunotoxin fusion proteinetc.). Suitable antibodies include, without limitation, antibodiesdirected against co-stimulatory or co-inhibitory molecules (CD28, CD40,CTLA-4, PD-1 etc.); and other therapeutic antibodies. Non-limitingexamples of suitable antibodies include, but are not limited to,adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab,bapineuzumab, basiliximab, belimumab, briakinumab, brodalumab,canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab,eculizumab, etrolizumab, gemtuzumab ozogamicin, golimumab, ibritumomabtiuxetan, labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab,muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab,oregovomab, palivizumab, panitumumab, pemtumornab, pertuzumab,ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab,trastuzumab, ustekinumab, vedolizomab, zalutumumab, and zanolimumab.

Non-limiting examples of therapeutic and prophylactic antibodies thatcan be used in combination therapy with an immunomodulatory compositionof the present disclosure include MDX-010 (Medarex, N.J.) which is ahumanized anti-CTLA-4 antibody for the treatment of prostate cancer;SYNAGIS™ (MedImmune, Md.) which is a humanized anti-respiratorysyncytial virus (RSV) monoclonal antibody for the treatment of RSVinfection; and HERCEPTIN™ (Trastuzumab) (Genentech, Calif.) which is ahumanized anti-HER2 monoclonal antibody for the treatment of metastaticbreast cancer. Other examples are humanized anti-CD18 F(ab′)₂(Genentech); CDP860 which is a humanized anti-CD18 F(ab′)₂ (Celltech,UK); PRO542 which is an anti-HIV gp120 antibody fused with CD4(Progenics/Genzyme Transgenics); Ostavir which is a human anti-HepatitisB virus antibody (Protein Design Lab/Novartis); PROTOVIR™ which is ahumanized anti-CMV IgGI antibody (Protein Design Lab/Novartis); MAK-195(SEGARD) which is a murine anti-TNF-α (Knoll Pharma/BASF); IC14 which isan anti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgG1 antibody(Genentech); OVAREX™ which is a murine anti-CA 125 antibody (Altarex);PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2aantibody (Glaxo Wellcome/Centocor); BEC2 which is a murine anti-idiotype(GD3 epitope) IgG antibody (ImClone System); IMC-C225 which is achimeric anti-EGFR IgG antibody (ImClone System); VITAXIN™ which is ahumanized anti-αVβ3 integrin antibody (Applied MolecularEvolution/MedImmune); Campath 1H/LDP-03 which is a humanized anti-CD52IgG1 antibody (Leukosite); Smart M195 which is a humanized anti-CD33 IgGantibody (Protein Design Lab/Kanebo); RITUXAN™ which is a chimericanti-CD20 IgG1 antibody (IDEC Pharm/Genentech, Roche/Zettyaku);LYMPHOCIDE™ which is a humanized anti-CD22 IgG antibody (Immunomedics);Smart ID10 which is a humanized anti-HLA antibody (Protein Design Lab);ONCOLYM™ (Lym-1) is a radiolabelled murine anti-HLA DIAGNOSTIC REAGENTantibody (Techniclone); ABX-IL8 is a human anti-IL8 antibody (Abgenix);anti-CD11a is a humanized IgG1 antibody (Genentech/Xoma); ICM3 is ahumanized anti-ICAM3 antibody (ICOS Pharm); IDEC-114 is a primatizedanti-CD80 antibody (IDEC Pharm/Mitsubishi); ZEVALIN™ is a radiolabelledmurine anti-CD20 antibody (IDEC/Schering AG); IDEC-131 is a humanizedanti-CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized anti-CD4antibody (IDEC); IDEC-152 is a primatized anti-CD23 antibody(IDEC/Seikagaku); SMART anti-CD3 is a humanized anti-CD3 IgG (ProteinDesign Lab); 5G1.1 is a humanized anti-complement factor 5 (C5) antibody(Alexion Pharm); D2E7 is a humanized anti-TNF-α antibody (CAT/BASF);CDP870 is a humanized anti-TNF-α Fab fragment (Celltech); IDEC-151 is aprimatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKline Beecham);MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571is a humanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4A isa humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™ is ahumanized anti-CD40L IgG antibody (Biogen); ANTEGREN™ is a humanizedanti-VLA-4 IgG antibody (Elan); MDX-33 is a human anti-CD64 (FcγR)antibody (Medarex/Centeon); SCH55700 is a humanized anti-IL-5 IgG4antibody (Celltech/Schering); SB-240563 and SB-240683 are humanizedanti-IL-5 and IL-4 antibodies, respectively, (SmithKline Beecham);rhuMab-E25 is a humanized anti-IgE IgG1 antibody(Genentech/Norvartis/Tanox Biosystems); ABX-CBL is a murine anti CD-147IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(MedImmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′).sub.2(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor). The above-listed immunoreactive reagents, as wellas any other immunoreactive reagents, may be administered according toany regimen known to those of skill in the art, including the regimensrecommended by the suppliers of the immunoreactive reagents.

A subject combination therapy can involve: administration of animmunomodulatory composition of the present disclosure and one or morecytokines. For example, in some embodiments, a subject method involves:a) administration of an immunomodulatory composition of the presentdisclosure; and b) administration of one or more cytokines. The HKCC andthe one or more cytokines can be in the same formulation or in separateformulations. The HKCC and the one or more cytokines can be administeredsimultaneously, or at different times. Suitable cytokines include,without limitation, interleukins, transforming growth factors (TGFs),fibroblast growth factors (FGFs), platelet derived growth factors(PDGFs), epidermal growth factors (EGFs), colony stimulating factors(CSFs), connective tissue activated peptides (CTAPs), osteogenicfactors, and biologically active analogs, fragments, and derivatives ofsuch growth factors. Suitable cytokines include B/T-cell differentiationfactors, B/T-cell growth factors, mitogenic cytokines, chemotacticcytokines, colony stimulating factors, angiogenesis factors, IFN-α,IFN-β, IFN-γ, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11,IL12, IL13, IL14, IL15, IL16, IL17, IL18, IL22, etc., leptin, myostatin,macrophage stimulating protein, platelet-derived growth factor, tumornecrosis factor (TNF)-alpha (TNF-α), TNF-β, nerve growth factor (NGF),CD40L, CD137L/4-1BBL, human lymphotoxin-β, G-CSF, M-CSF, GM-CSF,platelet-derived growth factor (PDGF), IL-1a, IL1-β, IP-10, PF4, GRO,9E3, erythropoietin, endostatin, angiostatin, vascular endothelialgrowth factor (VEGF) or any fragments or combinations thereof. Othercytokines include members of the transforming growth factor (TGF)supergene family include the beta transforming growth factors (forexample TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (forexample, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);heparin-binding growth factors (for example, fibroblast growth factor(FGF), epidermal growth factor (EGF), platelet-derived growth factor(PDGF), insulin-like growth factor (IGF)); hematopoietic growth factors(Flt3); pituitary growth hormones or derivatives; growth hormones,neuroactive hormones, Inhibins (for example, Inhibin A, Inhibin B);differentiation factors (for example, GDF-1); and Activins (for example,Activin A, Activin B, Activin AB).

A subject combination therapy can involve: administration of animmunomodulatory composition of the present disclosure and one or moretherapeutic agents such as anti-angiogenic agents (e.g., in methods forthe treatment of solid tumors and for the treatment and prevention ofmetastases) and anti-hormonal agents (particularly in methods for thetreatment of hormone-dependent cancers such as breast cancer andprostate cancer).

In one embodiment, an immunomodulatory composition of the presentdisclosure is administered in combination with one or moreanti-angiogenic agents. Such agents include, without limitation,angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine ProteaseInhibitor) anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminalproteolytic fragments of fibronectin, 16 kDa proteolytic fragment ofprolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a 13-aminoacid peptide corresponding to a fragment of platelet factor-4 (Maione etal., 1990, Cancer Res. 51:2077-2083), a 14-amino acid peptidecorresponding to a fragment of collagen I (Tolma et al., 1993, J. CellBiol. 122:497-511), a 19 amino acid peptide corresponding to a fragmentof Thrombospondin I (Tolsma et al., 1993, J. Cell Biol. 122:497-511), a20-amino acid peptide corresponding to a fragment of SPARC (Sage et al.,1995, J. Cell. Biochem. 57:1329-1334), or any fragments, family members,or variants thereof, including pharmaceutically acceptable saltsthereof.

Other peptides that inhibit angiogenesis and correspond to fragments oflaminin, fibronectin, procollagen, and EGF have also been described(see, e.g., Cao, 1998, Prog Mol Subcell Biol. 20:161-176). Monoclonalantibodies and cyclic pentapeptides, which block certain integrins thatbind RGD proteins (i.e., possess the peptide motif Arg-Gly-Asp), havebeen demonstrated to have anti-vascularization activities (Brooks etal., 1994, Science 264:569-571; Hammes et al., 1996, Nature Medicine2:529-533). Moreover, inhibition of the urokinase plasminogen activatorreceptor by receptor antagonists inhibits angiogenesis, tumor growth andmetastasis (Min et al., 1996, Cancer Res. 56: 2428-33; Crowley et al.,1993, Proc Natl Acad Sci. 90:5021-25).

In another embodiment, a combination therapy of the present disclosurecomprises administering an immunomodulatory composition of the presentdisclosure together with a hormonal treatment modality. Such treatmentmodalities include the administration of hormonal antagonists (e.g.,flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate(LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis andprocessing, and steroids (e.g., dexamethasone, retinoids, deltoids,betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone,glucocorticoids, mineralocorticoids, estrogen, testosterone,progestins), vitamin A derivatives (e.g., all-trans retinoic acid(ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone,onapristone), and antiandrogens (e.g., cyproterone acetate).

In another embodiment, an immunomodulatory composition of the presentdisclosure is used in association with a treatment modality thatutilizes polynucleotide compounds, such as antisense polynucleotides,ribozymes, RNA interference molecules, triple helix polynucleotides andthe like.

In certain embodiments, an immunomodulatory composition of the presentdisclosure is administered in combination with an immunoregulatoryagent. In some embodiments, the immunomodulatory composition isformulated with the immunoregulatory agent. An “immunoregulatory agent”is a substance that suppresses, masks, or enhances the immune system ofthe subject to whom it is administered. Exemplary agents are those thatsuppress cytokine production, downregulate or suppress self-antigenexpression, or mask the MHC antigens. Examples of such agents include2-amino-6-aryl-5-substituted pyrimidines (see, U.S. Pat. No. 4,665,077),azathioprine (or cyclophosphamide, if there is an adverse reaction toazathioprine); bromocryptine; glutaraldehyde (which masks the MHCantigens, as described in U.S. Pat. No. 4,120,649); anti-idiotypicantibodies for MHC antigens and MHC fragments; cyclosporin A; steroidssuch as glucocorticosteroids, e.g., prednisone, methylprednisolone, anddexamethasone; cytokine or cytokine receptor antagonists includinganti-interferon-γ, -β, or α antibodies; anti-tumor necrosis factor-aantibodies; anti-tumor necrosis factor-.beta. antibodies;anti-interleukin-2 antibodies and anti-IL-2 receptor antibodies;anti-L3T4 antibodies; heterologous anti-lymphocyte globulin; pan-Tantibodies, preferably anti-CD3 or anti-CD4/CD4a antibodies; solublepeptide containing a LFA-3 binding domain; streptokinase; TGF-β;streptodomase; FK506; RS-61443; deoxyspergualin; and rapamycin. Examplesof cytokines include, but are not limited to lymphokines, monokines, andtraditional polypeptide hormones. Included among the cytokines aregrowth hormone such as human growth hormone, N-methionyl human growthhormone, and bovine growth hormone; parathyroid hormone; thyroxine;insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such asfollicle stimulating hormone (FSH), thyroid stimulating hormone (TSH),and luteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoiotin (TPO); nerve growth factors such as NGF-α;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons; colony stimulating factors (CSFs)such as macrophage-CSF (M-CSF); granulocyte-macrophage-CgP (GM-CSP); andgranulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1a, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumornecrosis factor such as TNF-α or TNF-β; and other polypeptide factorsincluding LIF and kit ligand (KL). As used herein, the term cytokineincludes proteins from natural sources or from recombinant cell cultureand biologically active equivalents of the native sequence cytokines.

In certain embodiments, an immunomodulatory composition of the presentdisclosure is administered in combination therapy with one or moreimmunomodulatory agents, e.g., a cytokine. Suitable cytokines include,but are not limited to, interleukin-1 (IL-1), IL-2, IL-3, IL-12, IL-15,IL-18, G-CSF, GM-CSF, thrombopoietin, and γ interferon.

In certain embodiments, an immunomodulatory composition of the presentdisclosure is administered in combination with a compound that enhancesmonocyte or macrophase function. In certain embodiments, a compound thatenhances monocyte or macrophage function (e.g., at least about 25%, 50%,75%, 85%, 90%, 9% or more) can be used in conjunction with animmunomodulatory composition of the present disclosure. Such compoundsare known in the art and include, without limitation, cytokines such asinterleukins (e.g., IL-12), and interferons (e.g., alpha or gammainterferon). In certain embodiments, the compound that enhances monocyteor macrophage function is formulated with an immunomodulatorycomposition of the present disclosure and is thus administeredconcurrently with the immunomodulatory composition of the presentdisclosure. In other embodiments, the compound that enhances monocyte ormacrophage function is administered separately from the immunomodulatorycomposition of the present disclosure and can be administeredconcurrently (within a period of hours of each other), during the samecourse of therapy, or sequentially with the immunomodulatory compositionof the present disclosure. In some embodiments, the compound thatenhances monocyte or macrophage function is administered to a humansubject. In one embodiment, the human subject has a blood leukocyte,monocyte, neutrophil, lymphocyte, and/or basophil count that is withinthe normal range for humans. Normal ranges for human blood leukocytes(total) are about 3.5-10.5 (10⁹/L). Normal ranges for human bloodneutrophils are about 1.7-7.0 (10⁹/L), monocytes is about 0.3-0.9(10⁹/L), lymphocytes is about 0.9-2.9 (10⁹/L) basophils is about 0-0.3(10⁹/L), and eosinophils is about 0.05-0.5 (10⁹/L). In otherembodiments, the human subject has a blood leukocyte count that is lessthan the normal range for humans, for example at least about 0.01, 0.05,0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, or 0.8 (10⁹/L) leukocytes.

Methods of Enhancing an Anti-Bacterial Immune Response

The present disclosure provides methods of enhancing an immune responseto a bacterium or a substance produced by a bacterium, the methodcomprising administering to an individual in need thereof an effectiveamount of an immunomodulatory composition of the present disclosure. Insome cases, a method of the present disclosure of enhancing an immuneresponse to a bacterium or a substance produced by a bacterium comprisesadministering to an individual in need thereof an effective amount of animmunomodulatory composition of the present disclosure, where theimmunomodulatory composition comprises a bacterial antigen (other thanan antigen of HKCC). Suitable bacterial antigens are described above.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a bacterium, or a substance produced by a bacterium, iseffective to reduce the number of bacteria (e.g., pathogenic bacteria)in the individual by at least about 25%, at least about 50%, at leastabout 75%, or at least about 99%, compared to a pre-treatment number ofpathogenic bacteria in the individual, or to an extent that thepathogenic bacterium cannot be detected in the individual (e.g., in abiological sample obtained from the individual).

In some cases, a method of the present disclosure of enhancing an immuneresponse to a bacterium, or a substance produced by a bacterium, iseffective to induce or enhance an immune response to a pathogenicbacterium. Pathogenic bacteria include, e.g., Gram positive bacteria,Gram negative bacteria, mycobacteria, etc. Non-limiting examples ofpathogenic bacteria include Mycobacteria, Streptococcus, Staphylococcus,Pseudomonas, Salmonella, Neisseria, and Listeria. In some cases, thebacteria is Neisseria gonorrhea, M. tuberculosis, M. leprae, Listeriamonocytogenes, Streptococcus pneumoniae, S. pyogenes, S. agalactiae, S.viridans, S. faecalis, S. aureus, S. epidermis, or S. Bovis.

Other examples of pathogenic bacteria contemplated include, but are notlimited to, Gram positive bacteria (e.g., Listeria, Bacillus such asBacillus anthracis, Erysipelothrix species), Gram negative bacteria(e.g., Bartonella, Brucella, Burkholderia, Campylobacter, Enterobacter,Escherichia, Francisella, Hemophilus, Klebsiella, Morganella, Proteus,Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, andYersinia species), spirochete bacteria (e.g., Borrelia species includingBorrelia burgdorferi that causes Lyme disease), anaerobic bacteria(e.g., Actinomyces and Clostridium species), Gram positive and negativecoccal bacteria, Enterococcus species, Streptococcus species,Pneumococcus species, Staphylococcus species, Neisseria species.

Additional non-limiting examples of specific infectious bacteria includeCitrobacter, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophila, Mycobacteria avium, M. intracellulare, M. kansaii, M.gordonae, M. africanum, Staphylococcus aureus, Neisseria meningitidis,Haemophilus influenzae, Bacillus anthracis, Yersinia pestis,Corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasteurella multocida, Fusobacterium nucleatum,Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue,Leptospira, Rickettsia, Porphyromonas gingivalis, and Actinomycesisraelli.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a bacterium, or a substance produced by a bacterium,comprises administering an immunomodulatory composition to an individualin need thereof, and further comprising administering to the individualan effective amount of an anti-bacterial or an antimycobacterial agent.Anti-bacterial and anti-mycobacterial agents are known in the art andinclude, e.g., beta-lactam antibiotics, tetracyclines, streptomycin,chloramphenicol, neomycin, gramicidin, bacitracin, sulfonamides,nitrofurazone, nalidixic acid, rifampicin, fluoroquinolones, isoniazid,pyrazinamide, vancomycin, methicillin etc.

Suitable anti-bacterial agents include, e g, Aminoglycosides such asAmikacin, Apramycin, Arbekacin, Bambermycins, Butirosin, Dibekacin,Dihdrostreptomycin, Fortimicin(s), Gentamicin, Ispamicin, Kanamycin,Micronomicin, Neomycin, Neomycin Undecylenate, Netilmicin, Paromomycin,Ribostamycin, Sisomicin, Spectinomycin, Streptomycin, Streptonicozid andTobramycin; Ansamycins such as Rifamide, Rifampin, Rifamycin andRifaximin; β-lactams such as Carbapenems such as Imipenem;Cephalosporins such as Cefactor, Cefadroxil, Cefamandole, Cefatrizine,Cefazedone, Cefazolin, Cefixime, Cefinenoxime, Cefodizime, Cefonicid,Cefoperazone, Ceforanide, Cefotaxime, Cefotiam, Cefpimizole,Cefpirimide, Cefpodoxime Proxetil, Cefroxadine, Cefsulodin, Ceftazidime,Cefteram, Ceftezole, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefuroxime,Cefuzonam, Cephacetrile Sodium, Cephalexin, Cephaloglycin,Cephaloridine, Cephalosporin, Cephalothin, Cephapirin Sodium, Cephradineand Pivcefalexin; Cephamycins such as Cefbuperazone, Cefmetazole,Cefminox, Cefetan and Cefoxitin; Monobactams such as Aztreonam,Carumonam and Tigemonam; Oxacephems such as Flomoxef and Moxolactam;Penicillins such as Amidinocillin, Amdinocillin Pivoxil, Amoxicillin,Ampicillan, Apalcillin, Aspoxicillin, Azidocillan, Azlocillan,Bacampicillin, Benzylpenicillinic Acid, Benzylpenicillin Sodium,Carbenicillin, Carfecillin Sodium, Carindacillin, Clometocillin,Cloxacillin, Cyclacillin, Dicloxacillin, Diphenicillin Sodium,Epicillin, Fenbenicillin, Floxicillin, Hetacillin, Lenampicillin,Metampicillin, Methicillin Sodium, Mezlocillin, Nafcillin Sodium,Oxacillin, Penamecillin, Penethamate Hydriodide, Penicillin GBenethamine, Penicillin G Benzathine, Penicillin G Benzhydrylamine,Penicillin G Calcium, Penicillin G Hydrabamine, Penicillin G Potassium,Penicillin G Procaine, Penicillen N, Penicillin O, Penicillin V,Penicillin V Benzathine, Penicillin V Hydrabamine, Penimepicycline,Phenethicillin Potassium, Piperacillin, Pivapicillin, Propicillin,Quinacillin, Sulbenicillin, Talampicillin, Temocillin and Ticarcillin;Lincosamides such as Clindamycin and Lincomycin; Macrolides such asAzithromycin, Carbomycin, Clarithromycin, Erythromycin, ErythromycinAcistrate, Erythromycin Estolate, Erythromycin Glucoheptonate,Erythromycin Lactobionate, Erythromycin Propionate, ErythromycinStearate, Josamycin, Leucomycins, Midecamycins, Miokamycin,Oleandomycin, Primycin, Rokitamycin, Rosaramicin, Roxithromycin,Spiramycin and Troleandomycin; Polypeptides such as Amphomycin,Bacitracin, Capreomycin, Colistin, Enduracidin, Enviomycin, Fusafungine,Gramicidin(s), Gramicidin S, Mikamycin, Polymyxin, PolymyxinB-Methanesulfonic Acid, Pristinamycin, Ristocetin, Teicoplanin,Thiostrepton, Tuberactinomycin, Tyrocidine, Tyrothricin, Vancomycin,Viomycin, Viomycin Pantothenate, Virginiamycin and Zinc Bacitracin;Tetracyclines such as Apicycline, Chlortetracycline, Clomocycline,Demeclocycline, Doxycycline, Guamecycline, Lymecycline, Meclocycline,Methacycline, Minocycline, Oxytetracycline, Penimepicycline,Pipacycline, Rolitetracycline, Sancycline, Senociclin and Tetracycline;Cycloserine; Mupirocin; and Tuberin. Suitable anti-bacterial agentsinclude antibodies specific for a bacterium.

Methods of Enhancing an Anti-Viral Immune Response

The present disclosure provides methods of enhancing an immune responseto a virus, the method comprising administering to an individual in needthereof an effective amount of an immunomodulatory composition of thepresent disclosure. In some cases, a method of the present disclosure ofenhancing an immune response to a virus comprises administering to anindividual in need thereof an effective amount of an immunomodulatorycomposition of the present disclosure, where the immunomodulatorycomposition comprises a viral antigen. Suitable viral antigens aredescribed above.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a virus is effective to reduce the number of viruses (e.g.,pathogenic viruses) in the individual by at least about 25%, at leastabout 50%, at least about 75%, or at least about 99%, or to an extentthat the pathogenic virus cannot be detected in the individual (e.g., ina biological sample obtained from the individual).

For example, in some cases, a method of the present disclosure ofenhancing an immune response to a virus is effective to reduce the viralload in the individual by at least about 25%, at least about 50%, atleast about 75%, or at least about 99%, or to an extent that thepathogenic virus cannot be detected in the individual (e.g., in abiological sample obtained from the individual). In some cases, a methodof the present disclosure of enhancing an immune response to a virus iseffective to reduce the number of genome copies of the virus in theindividual by at least about 25%, at least about 50%, at least about75%, or at least about 99%, compared to a pre-treatment number of genomecopies of the virus in the individual, or to an extent that no genomecopies of the virus can be detected in the individual (e.g., in abiological sample obtained from the individual).

In some cases, a method of the present disclosure of enhancing an immuneresponse to a virus induces or increases an immune response to apathogenic virus. Pathogenic viruses include, but are not limited to,herpes viruses (HSV-1, HSV-2, VZV, EBV, CMV, HHV-6, HHV-8), influenzaviruses (Flu A, B), hepatitis viruses (HepA, HepB, HepC, HepD, HepE),human immunodeficiency viruses (HIV-1, HIV-2), respiratory syncytialviruses, measles viruses, rhinoviruses, adenoviruses, SARS viruses,papillomaviruses, orthopoxviruses, West Nile viruses, and a dengueviruses. Pathogenic viruses include members of the Flaviviridae familyof viruses. Pathogenic viruses include a flavivirus selected from thegroup consisting of dengue, Kunjin, Japanese encephalitits, West Nile,and yellow fever virus. Pathogenic viruses include lymphocyticchoriomenignitis virus, hepatitis B virus, Epstein Barr virus, and humanimmunodeficiency virus. Pathogenic viruses include, but are not limitedto: Retroviridae (e.g. human immunodeficiency viruses, such as HIV-1,also referred to as LAV or HTLV-III/LAV, or HIV-III; and other isolates,such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae(e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g.dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g. ebola-likeviruses; Marburg virus); Paramyxoviridae (e.g. parainfluenza viruses,mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaanviruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae(hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviursesand rotaviruses); Bornaviridae; Hepadnaviridae (Hepatitis B virus);Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyomaviruses); Adenoviridae (e.g., adenoviruses); Herpesviridae (herpessimplex virus (HSV) 1 and 2), varicella zoster virus, cytomegalovirus(CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, poxviruses); and Iridoviridae (e.g. African swine fever virus); andunclassified viruses (e.g. the etiological agents of Spongiformencephalopathies, the agent of delta hepatitis, thought to be adefective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1, internally transmitted; class 2, parenterallytransmitted, i.e., Hepatitis C Virus); Norwalk and related viruses, andastroviruses.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a virus, comprises administering an immunomodulatorycomposition to an individual in need thereof, and further comprisingadministering to the individual an effective amount of at least oneadditional therapeutic agent, e.g., an anti-viral agent.

Anti-viral agents are known in the art and include, e.g., an anti-HCVagent such as ribavirin and its analogues; glycosidase inhibitors;glucosidase inhibitors; IRES (internal ribosomal entry site), p7, entry,fusion, helicase, assembly, egress, NS2, NS3, NS4, NS5a and NS5Binhibitors; inosine monophosphate dehydrogenase inhibitors; cyclophilininhibitors; metalloprotease inhibitors; anti-HCV nucleos(t)ide andnon-nucleoside RNA polymerase inhibitors etc.; an anti-HIV agent;anti-HBV agent; and the like.

In some embodiments, the at least one additional therapeutic agent is aninterferon (e.g., interferon-alpha, interferon-beta, interferon-gamma,interferon-lambda, interferon-tau, interferon-omega, etc.). In someembodiments, the at least one additional therapeutic agent is IFN-α. Insome embodiments, the at least one additional therapeutic agent isIFN-β.

Suitable additional anti-viral agents for treating an HCV infectioninclude, but are not limited to, ribavirin and its prodrugs such asviramidine, telaprevir, sofosbuvir, boceprevir, ciluprevir, simeprevir,danoprevir, vaniprevir, MK-5172, MK-0608, 2′-C-methyl-7-deaza adenosine,2′-C-methyl-adenosines, BI201335, narlaprevir, asunaprevir, GS-9256,GS-9451, ABT-450, IDX-320, ACH-1625, Valopicitabine, mericitabine,R1626, PSI-938, INX-189, BILN1941, BI-207127, VCH222, VX-135, ANA598,ANA773, ABT-072, ABT-333, HCV-796, GS-9190, Daclatasavir, BMS-824393,BMS-791325, PPI-461, GS-5885, alisporivir (Debio-025), NIM-811, SCY-635,nitazoxanide, clemizole, miravirasen, celgosivir, BCX-5191, GSK-2336805,anti-PD-1 antibodies (CT-011), bavituximab (anti-phosphatidyl serineMab), therapeutic vaccine (GI-5005, IC-41, TG-4040) prophylactic vaccine(such as HCV E1/E2/MF-59), and the prodrugs thereof. Suitable additionaltherapeutic agents include, e.g., therapeutic agents for the treatmentof an hepatitis B virus infection include, but are not limited tolamivudine, adefovir, entecavir, telbuvudine, tenofovir and the prodrugsthereof.

For example, suitable additional anti-viral agents for treating an HCVinfection include weekly injections of pegylated IFN-α combined withtwice-daily oral doses of ribavirin(1-β-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide).

Suitable additional therapeutic agents include, e.g., therapeutic agentsfor the treatment of an immunodeficiency virus infection, or for thetreatment of a disorder that may accompany an immunodeficiency virusinfection (e.g., a bacterial infection, a fungal infection, and thelike). Suitable additional therapeutic agents include, e.g., beta-lactamantibiotics, tetracyclines, chloramphenicol, neomycin, gramicidin,bacitracin, sulfonamides, nitrofurazone, nalidixic acid, cortisone,hydrocortisone, betamethasone, dexamethasone, fluocortolone,prednisolone, triamcinolone, indomethacin, sulindac, acyclovir,amantadine, rimantadine, recombinant soluble CD4 (rsCD4), cyanovirin-N,microvirin, fuzeon, anti-receptor antibodies (e.g., for rhinoviruses),nevirapine, cidofovir (Vistide™), trisodium phosphonoformate(Foscarnet™), famcyclovir, pencyclovir, valacyclovir, nucleicacid/replication inhibitors, interferon, zidovudine (AZT, Retrovir™),didanosine (dideoxyinosine, ddl, Videx™), stavudine (d4T, Zerit™),zalcitabine (dideoxycytosine, ddC, Hivid™), nevirapine (Viramune™),lamivudine (Epivir™, 3TC), protease inhibitors, saquinavir (Invirase™,Fortovase™), ritonavir (Norvir™), nelfinavir (Viracept™), efavirenz(Sustiva™), abacavir (Ziagen™), amprenavir (Agenerase™) indinavir(Crixivan™), ganciclovir, AzDU, delavirdine (Rescriptor™), kaletra,trizivir, rifampin, clathiromycin, erythropoietin, colony stimulatingfactors (G-CSF and GM-CSF), non-nucleoside reverse transcriptaseinhibitors, nucleoside inhibitors, viral entry inhibitors, fusioninhibitors, integrase inhibitors, adriamycin, fluorouracil,methotrexate, asparaginase and combinations thereof. Additional suitabletherapeutic agents for HIV include integrase and fusion inhibitors suchas Raltegravir, Elvitegravir, Enfuvirtide, Maraviroc etc.

In some embodiments, the at least one additional therapeutic agent is aneuraminidase inhibitor, e.g., where the influenza virus is influenza Aor influenza B. Suitable neuraminidase inhibitors include, e.g.,oseltamivir (ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)cyclohex-1-ene-1-carboxylate;Tamiflu™), zanamivir(2R,3R,4S)-4-[(diaminomethylidene)amino]-3-acetamido-2-[(1R,2R-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylicacid; Relenza™), and peramivir(1S,2S,3S,4R)-3-[(1S)-1-acetanaido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid). In some embodiments, the at least one additional therapeuticagent is an M2 blocker, e.g., blocks a viral ion channel (M2 protein).The antiviral drugs amantadine and rimantadine are M2 blockers, and canbe used in subject method.

Suitable additional therapeutic agents, e.g., for the treatment of anHSV-1 or an HSV-2 infection include, but are not limited to, acyclovir(Zovirax), valganciclovir, famciclovir, valacyclovir (Valtrex),ganciclovir (Cytovene), cidofovir (Vistide), antisense oligonucleotidefomivirsen (Vitravene), foscarnet (Foscavir), penciclovir, idoxuridine,vidarabine, and trifluridine.

In some embodiments, the one or more different therapeutic agent isselected antiviral agents that target two or more different viruses;e.g., an HIV inhibitor, HBV inhibitor, HCV inhibitor, herpes virusinhibitor, influenza virus inhibitor, RNA inhibitor, interfering RNA(RNAi) inhibitor, natural products etc. In some cases, a method of thepresent disclosure of treating a viral infection comprises administeringan immunomodulatory composition to an individual in need thereof, andfurther comprising administering to the individual an effective amountof at least one additional therapeutic agent, e.g., a monoclonalantibody or antibody products directed against viral antigens, wheresuitable monoclonal antibodies include but are not limited to HBIg,antibodies against influenza virus strains, anti-hepatitis A virusantibody, SYNAGIS (anti-RSV Mab), anti-rabies antibody, ostavir(anti-HBV Mab), Pro542 (anti-HIV gp120), Potovir (anti-CMV Mab),anti-PD-1 antibodies (CT-011), bavituximab (anti-phosphatidyl serineMab) etc.

Methods of Enhancing an Immune Response to a Parasitic Infection

The present disclosure provides methods of enhancing an immune responseto a microbial parasite (e.g., a pathogenic protozoan; a helminth;etc.), the method comprising administering to an individual in needthereof an effective amount of an immunomodulatory composition of thepresent disclosure. In some cases, a method of the present disclosure ofenhancing an immune response to a microbial parasite comprisesadministering to an individual in need thereof an effective amount of animmunomodulatory composition of the present disclosure, where theimmunomodulatory composition comprises an antigen derived from amicrobial parasite (e.g., a protozoan antigen; a helminth antigen).Suitable microbial parasite antigens are described above.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a microbial parasite is effective to reduce the number ofmicrobial parasites (e.g., pathogenic protozoa; pathogenic helminths) inthe individual by at least about 25%, at least about 50%, at least about75%, or at least about 99%, compared to a pre-treatment number ofmicrobial parasite in the individual, or to an extent that the microbialparasite cannot be detected in the individual (e.g., in a biologicalsample obtained from the individual).

In some cases, a method of the present disclosure of enhancing an immuneresponse to a microbial parasite comprises administering animmunomodulatory composition to an individual in need thereof, andfurther comprising administering to the individual an effective amountof a least one additional therapeutic agent. Anti-parasitic agents areknown in the art and include, e.g., chloroquine, etc. For example,anti-malarial agents include, e.g., quinine, chloroquine, atovaquone,proguanil, primaquine, amodiaquine, mefloquine, piperaquine,artemisinin, methylene blue, pyrimethamine, sulfadoxine,artemether-lumefantrine, dapsone-chlorproguanil, artesunate, quinidine,clopidol, pyridine/pyridinol analogs, 4(1H)-quinolone analogs,dihydroartemisinin, a mixture of atovaquone and proguanil, anendoperoxide, and an acridone. Anti-parasitic agents include antibodiesspecific for the parasite.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a microbial parasite induces or increases an immune responseto a microbial parasite such as Plasmodium spp., Toxoplasma gondii,Babesia spp., Trichinella spiralis, Entamoeba histolytica, Giardialamblia, Enterocytozoon bieneusi, Naegleria, Acanthamoeba, Trypanosomarhodesiense and Trypanosoma gambiense, Isospora spp., Cryptosporidiumspp, Eimeria spp., Neospora spp., Sarcocystis spp., and Schistosoma spp.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a protozoan parasite induces or increases an immune responseto a protozoan parasite such as Giardia; a plasmodium species (e.g.,Plasmodium falciparum); Toxoplasma gondii; a cryptosporidium; aTrichomonas species; a trypanosome (e.g., Trypanosoma cruzi); orLeishmania.

Methods of Enhancing an Immune Response to a Pathogenic Fungus

The present disclosure provides methods of enhancing an immune responseto a pathogenic fungus, the method comprising administering to anindividual in need thereof an effective amount of an immunomodulatorycomposition of the present disclosure. In some cases, a method of thepresent disclosure of enhancing an immune response to a pathogenicfungus comprises administering to an individual in need thereof aneffective amount of an immunomodulatory composition of the presentdisclosure, where the immunomodulatory composition comprises an antigenderived from a pathogenic fungus. Suitable fungal antigens are describedabove.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a pathogenic fungus is effective to reduce the number offungal bodies in the individual by at least about 25%, at least about50%, at least about 75%, or at least about 99%, compared to apre-treatment number of fungal bodies in the individual, or to an extentthat the pathogenic fungus cannot be detected in the individual (e.g.,in a biological sample obtained from the individual).

In some cases, a method of the present disclosure of enhancing an immuneresponse to a pathogenic fungus induces or increases an immune responseto a fungus such as Candida spp. including C. albicans, Aspergillusspp., Cryptococcus spp. including C. neoformans, Blastomyces sp.,Pneumocytes spp., or Coccidioides spp.

In some cases, a method of the present disclosure of enhancing an immuneresponse to a pathogenic fungus comprises administering animmunomodulatory composition to an individual in need thereof, andfurther comprising administering to the individual an effective amountof a least one additional therapeutic agent. Anti-fungal agents areknown in the art and include, e.g., flucanazole, 5-fluorocytosine, etc.

Suitable anti-fungal agents include, e.g., Polyenes such asAmphotericin-B (including various formulations of Amphotericin-B),Candicidin, Dermostatin, Filipin, Fungichromin, Hachimycin, Hamycin,Lucensomycin, Mepartricin, Natamycin, Nystatin, Pecilocin and Perimycin;and others such as Azaserine, Griseofulvin, Oligomycins, NeomycinUndecylenate, Pyrrolnitrin, Siccanin, Tubercidin and Viridin;Allylamines such as Naftifine and Terbinafine; Imidazoles such asBifonazole, Butoconazole, Chlordantoin, Chlormidazole, Cloconazole,Clotrimazole, Econazole, Enilconazole, Fenticonazole, Isoconazole,Ketoconazole, Miconazole, Omoconazole, Oxiconazole, Nitrate, Sulconazoleand Tioconazole; Triazoles such as Fluconazole, Itraconazole andTerconazole; and other others such as Acrisorcin, Amorolfine,Biphenamine, Bromosalicylchloranilide, Buclosamide, Calcium Propionate,Chlophenesin, Ciclopirox, Cloxyquin, Coparaffinate, Diamthazole,Dihydrochloride, Exalamide, Flucytosine, Halethazole, Hexetidine,Loflucarban, Nifuratel, Potassium Iodide, Propionic Acid, Pyrithione,Salicylanilide, Sodium Propionate, Sulbentine, Tenonitrozole,Tolciclate, Tolindate, Tolnaftate, Tricetin, Ujothion, Undecylenic Acidand Zinc Propionate.

Methods of Treating an Allergic Disease

The present disclosure provides methods of treating an allergic diseasein an individual, the method comprising administering to the individualan effective amount of an immunomodulatory composition of the presentdisclosure. In some cases, a method of the present disclosure oftreating an allergic disease comprises administering to an individual inneed thereof an effective amount of an immunomodulatory composition ofthe present disclosure, where the immunomodulatory composition comprisesan allergen. Suitable allergens are described above.

In some cases, a subject a method of the present disclosure of treatingan allergic disease is effective to shift an immune response from a Th2immune response to a Th1 immune response and/or regulate an immuneresponse. In some cases, a subject a method of the present disclosure oftreating an allergic disease is effective to decrease one or more of: a)the level of IgE in an individual; b) the level of allergen-specific IgEin an individual; c) the number of mast cells in the individual; d) thelevel of histamine in the individual; e) the level of a Th2-associatedcytokine in the individual; f) a Th2 immune response; and g) the levelof IL-4 in the individual, compared to a pre-treatment level.

In some cases, a method of the present disclosure of treating anallergic disease comprises administering an immunomodulatory compositionto an individual in need thereof, and further comprising administeringto the individual an effective amount of at least one additionaltherapeutic agent. Suitable additional therapeutic agents include, e.g.,anti-histamines, steroids (e.g., corticosteroids), prostaglandininducers, anti-inflammatory agents, leukotriene antagonists, 1L-4muteins, soluble 1L-4 receptors, immunosuppressants (such as tolerizingpeptide vaccine), anti-IL-4 antibodies, IL-4 antagonists, anti-IL-5antibodies, soluble IL-13 receptor-Fc fusion proteins, anti-1L-9antibodies, CCR3 antagonists, CCR5 antagonists, VLA-4 inhibitors, anddownregulators of IgE. Suitable steroids include, but are not limitedto, beclomethasone, fluticasone, tramcinolone, budesonide,corticosteroids and budesonide.

Methods of Treating Cancer

The present disclosure provides methods of treating cancer in anindividual, the method comprising administering to the individual aneffective amount of an immunomodulatory composition of the presentdisclosure. In some cases, a method of the present disclosure fortreating cancer involves treating cancer located in a tissue, treatingcancer located an organ, or treating a metastatically spread cancer. Insome cases, a method of the present disclosure of treating cancercomprises administering to an individual in need thereof an effectiveamount of an immunomodulatory composition of the present disclosure,where the immunomodulatory composition comprises an tumor-associatedantigen. Suitable tumor-associated antigens are described above.

In some cases, a method of the present disclosure of treating cancer issuitable for treating a cancer selected from leukemia, acute leukemia,acute lymphocytic leukemia, acute myelocytic leukemia, myeloblastic,promyelocytic, myelomonocytic, monocytic, erythroleukemia, chronicleukemia, chronic myelocytic, (granulocytic) leukemia, chroniclymphocytic leukemia, Polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's disease, Multiple myeloma, Waldenstrom'smacroglobulinemia, Heavy chain disease, solid tumors, sarcomas andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, neuroblastoma sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, brain tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterinecancer, reproductive tract cancer, colorectal cancer, vulvar cancer,testicular tumor, lung carcinoma, small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, keratoacanthoma, andneuroblastomaretinoblastoma, Kaposi sarcoma, cutaneous lymphoma andmetastases.

In some cases, a subject a method of the present disclosure of treatinga cancer is effective to reduce the number of cancer cells in anindividual by at least about 25%, at least about 50%, at least about75%, or at least about 99% compared to a pre-treatment number of cancercells, or to an extent that the cancer cannot be detected in theindividual (e.g., in a biological sample obtained from the individual).

In some cases, a subject a method of the present disclosure of treatinga cancer is effective to increase survival and inhibit tumor growth inan individual. For example, in some cases, a subject a method of thepresent disclosure of treating a cancer is effective to increasesurvival by at least about 5%, at least about 10%, at least about 20%,at least about 25%, or more than 25%, compared to survival in theabsence of treatment with a method of the present disclosure.

In some cases, a method of the present disclosure of treating a cancercomprises administering an immunomodulatory composition to an individualin need thereof, and further comprising administering to the individualan effective amount of a least one additional therapeutic agent, e.g., acancer chemotherapeutic agent.

Chemotherapeutic agents are compounds that reduce proliferation ofcancer cells, and encompass cytotoxic agents and cytostatic agents.Non-limiting examples of chemotherapeutic agents include alkylatingagents, nitrosoureas, antimetabolites, antitumor antibiotics, plant(Vinca) alkaloids, hypoxic agents, and steroid hormones.

Agents that act to reduce cellular proliferation are known in the artand widely used. Such agents include alkylating agents, such as nitrogenmustards, nitrosoureas, ethylenimine derivatives, alkyl sulfonates, andtriazenes, including, but not limited to, mechlorethamine,cyclophosphamide (Cytoxan™), melphalan (L-sarcolysin), carmustine(BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin,chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil,pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan,dacarbazine, and temozolomide.

Antimetabolite agents include folic acid analogs, pyrimidine analogs,purine analogs, and adenosine deaminase inhibitors, including, but notlimited to, cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil(5-FU), floxuridine (FudR), 6-thioguanine, 6-mercaptopurine (6-MP),pentostatin, 5-fluorouracil (5-FU), methotrexate,10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, fludarabinephosphate, pentostatin, gemcitabine, cyclocytidine, guanazole, inosineglycodialdehyde, EICAR, ribavirin, tiazofurin, defroxamine andpyrazoloimidazole.

Suitable natural products and their derivatives, (e.g., vinca alkaloids,antitumor antibiotics, enzymes, lymphokines, and epipodophyllotoxins),include, but are not limited to, Ara-C, paclitaxel (Taxol®), docetaxel(Taxotere®), deoxycoformycin, mitomycin-C, L-asparaginase, azathioprine;brequinar; alkaloids, e.g. vincristine, vinblastine, vinorelbine,vindesine, etc.; podophyllotoxins, e.g. etoposide, teniposide,camptothecin etc.; antibiotics, e.g. anthracycline, daunorubicinhydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin,doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizonebiscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g.bleomycin; anthraquinone glycosides, e.g. plicamycin (mithramycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; macrocyclic immunosuppressants, e.g. cyclosporine, FK-506(tacrolimus, prograf), rapamycin, etc.; antivascular flavonoids; and thelike. Other agents include minerals, nutrients, vitamins, supplements,anti-oxidants, and anti-inflammatory treatments and modalities.

Other anti-proliferative cytotoxic agents are navelbene, CPT-11,anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide,folic acid, retinoic acid, ifosamide, and droloxafine. Other suitableanti-proliferative agents include siRNA, interfering RNA (RNAi), andanti-sense RNA.

Microtubule affecting agents that have antiproliferative activity arealso suitable for use and include, but are not limited to,allocolchicine (NSC 406042), Halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolstatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®), Taxol® derivatives, docetaxel (Taxotere®), thiocolchicine (NSC361792), trityl cysterin, vinblastine sulfate, vincristine sulfate,natural and synthetic epothilones including but not limited to,eopthilone A, epothilone B, discodermolide; estramustine, nocodazole,and the like.

Hormone modulators and steroids (including synthetic analogs) that aresuitable for use include, but are not limited to, adrenocorticosteroids,e.g. prednisone, dexamethasone, etc.; estrogens and pregestins, e.g.hydroxyprogesterone caproate, medroxyprogesterone acetate, megestrolacetate, estradiol, clomiphene, tamoxifen; etc.; and adrenocorticalsuppressants, e.g. aminoglutethimide; 17α-ethinylestradiol;diethylstilbestrol, testosterone, fluoxymesterone, dromostanolonepropionate, testolactone, methylprednisolone, methyl-testosterone,prednisolone, triamcinolone, chlorotrianisene, hydroxyprogesterone,aminoglutethimide, estramustine, medroxyprogesterone acetate,leuprolide, Flutamide (Drogenil), Toremifene (Fareston), and Zoladex®.Estrogens stimulate proliferation and differentiation; therefore,compounds that bind to the estrogen receptor are used to block thisactivity. Corticosteroids may inhibit T cell proliferation.

Other chemotherapeutic agents include metal complexes, e.g. cisplatin(cis-DDP), carboplatin, etc.; ureas, e.g. hydroxyurea; and hydrazines,e.g. N-methylhydrazine; epidophyllotoxin; a topoisomerase inhibitor;procarbazine; mitoxantrone; leucovorin; tegafur; etc. Otheranti-proliferative agents of interest include immunosuppressants, e.g.mycophenolic acid, thalidomide, desoxyspergualin, azasporine,leflunomide, mizoribine, azaspirane (SKF 105685); Iressa® (ZD 1839,4-(3-chloro-4-fluorophenylamino)-7-methoxy-6-(3-(4-morpholinyl)propoxy)quinazoline);etc.

“Taxanes” include paclitaxel, as well as any active taxane derivative orpro-drug. “Paclitaxel” (which should be understood herein to includeanalogues, formulations, and derivatives such as, for example,docetaxel, TAXOL™, TAXOTERE™ (a formulation of docetaxel), 10-desacetylanalogs of paclitaxel and 3′N-desbenzoyl-3′N-t-butoxycarbonyl analogs ofpaclitaxel) may be readily prepared utilizing techniques known to thoseskilled in the art (see also WO 94/07882, WO 94/07881, WO 94/07880, WO94/07876, WO 93/23555, WO 93/10076; U.S. Pat. Nos. 5,294,637; 5,283,253;5,279,949; 5,274,137; 5,202,448; 5,200,534; 5,229,529; and EP 590,267),or obtained from a variety of commercial sources, including for example,Sigma Chemical Co., St. Louis, Mo. (T7402 from Taxus brevifolia; orT-1912 from Taxus yannanensis).

Biological response modifiers suitable for use in connection with themethods of the present disclosure include, but are not limited to, (1)inhibitors of tyrosine kinase (RTK) activity; (2) inhibitors ofserine/threonine kinase activity; (3) tumor-associated antigenantagonists, such as antibodies that bind specifically to a tumorantigen; (4) apoptosis receptor agonists; (5) interleukin-2; (6)interferon-α.; (7) interferon-γ; (8) colony-stimulating factors; (9)inhibitors of angiogenesis; (10) antagonists of tumor necrosis factor;and (11) BRAF inhibitors.

In some cases, a method of the present disclosure of treating a cancercomprises administering an immunomodulatory composition to an individualin need thereof, and further comprising administering to the individualan effective amount of at least one additional therapeutic agent, e.g.,a monoclonal antibody directed against cancer antigens, where suitablemonoclonal antibodies include, but are not limited to, trastuzumab(Herceptin), bevacizumab (Avastin™), rituximab (Rituxan), Oregovomab,Lambrolizumab, Ipilimumab, pertuzumab, ranibizumab (Lucentis™),Cetuximab^(R), Camptosar^(R), Erbitux, Brevarex, Ovarex, Pentorex etc.;antibody directed against negative receptors such as PDl and CTLA-4;antibody directed against co-stimulatory receptors e.g., CD134 andCD137; CDP-860 (anti-CD18), antibody directed against cytokines such asIL-10 and TGF-b, and the like.

In some embodiments, the one or more different therapeutic agent isselected from different categories of anticancer agents describedherein.

In some cases, a method of the present disclosure of treating a cancercomprises enhancing recovery of an individual undergoing or havingundergone cancer therapy e.g., chemotherapy, radiation therapy, lasertherapy, therapeutic vaccine therapy, surgical resection etc. In somecases, a method of the present disclosure of treating cancer comprises,in addition to administering an immunogenic composition of the presentdisclosure, administering live, killed or attenuated microbial pathogenssuch as bacterial cells (e.g., S. pyogenes, S. aureus), or viruses(e.g., pox viruses, herpes viruses, measles viruses, vaccinia viruses,rotaviruses, oncolytic viruses etc.).

In some cases, a method of the present disclosure of treating a cancercomprises administering an immunomodulatory composition to an individualin need thereof, and further comprising administering to the individualan effective amount of at least one additional vaccine (e.g., BCGvaccine, measles vaccine, rotavirus vaccine etc.).

Methods of Treating an Autoimmune Disorder

The present disclosure provides methods of treating an autoimmunedisorder in an individual, the method comprising administering to theindividual an effective amount of an immunomodulatory composition of thepresent disclosure. Autoimmune conditions account for many autoimmunedisorders such as rheumatoid arthritis, asthma, type 1 diabetes,systemic lupus erythrymetosus (SLE), atherosclerosis, autoimmunehepatitis, celiac disease, autoimmune hemolytic anemia, etc. Bymodulating innate and adaptive immune mechanisms through theimmunomodulatory composition of the present disclosure, autoimmunedisorders can be treated. In some cases, a method of the presentdisclosure of treating an autoimmune disorder comprises administering toan individual in need thereof an effective amount of an immunomodulatorycomposition of the present disclosure, where the immunomodulatorycomposition comprises an autoantigen. Suitable autoantigens aredescribed above.

In some cases, a subject a method of the present disclosure of treatingan autoimmune disorder is effective to reduce the number and/or activityof self-reactive T cells in an individual by at least about 25%, atleast about 50%, at least about 75%, or at least about 99% compared to apre-treatment number and/or activity of self-reactive T cells, or to anextent that self-reactive T cells cannot be detected in the individual(e.g., in a biological sample obtained from the individual).

In some cases, a subject a method of the present disclosure of treatingan autoimmune disorder is effective to reduce the level ofautoantibodies in an individual by at least about 25%, at least about50%, at least about 75%, or at least about 99% compared to apre-treatment level of autoantibodies, or to an extent thatautoantibodies cannot be detected in the individual (e.g., in abiological sample obtained from the individual).

In some cases, a method of the present disclosure of treating anautoimmune disorder comprises administering an immunomodulatorycomposition to an individual in need thereof, and further comprisingadministering to the individual an effective amount of a least oneadditional therapeutic agent. Examples of therapeutic agents that can beused to treat autoimmune disorders include, but are not limited to,anti-inflammatory agents; immunosuppressive agents (e.g.,corticosteroids (e.g., prednisone, cortisol, methylprednisolone, etc.)),cyclosporin A); cytotoxic agents (e.g., 6-mercaptopurine, azathioprine,methotrexate, alkylating agents); danazol; colchisine; levamisole; andthe like.

Methods of Treating Diseases Comprising an Immune Dysregulation

The present disclosure provides methods of modulating and/or regulatingan immune dysfunction in an individual, the method comprisingadministering to the individual an effective amount of animmunomodulatory composition of the present disclosure. Immunedysfunction conditions account for many diseases such as rheumatoidarthritis, diabetes, psoriasis, systemic lupus erythematosus,graft-versus-host disease (GVHD), colitis, Crohn's disease, Alopeciaareata, asthma, allergic rhinitis, conjunctivitis, transplant rejection,Hashimoto's thyroiditis, inflammatory bowel diseases, cardiovasculardiseases, obesity, wound healing, burn recovery, aging, etc. Bymodulating innate and adaptive immune mechanisms through theimmunomodulatory composition of the present disclosure, immunedysfunction disorders can be prevented and/or treated.

In some cases, a method of the present disclosure of treating an immunedysfunction disorder comprises administering an immunomodulatorycomposition to an individual in need thereof, and further comprisingadministering to the individual an effective amount of at least oneadditional therapeutic agent.

In some cases, the method comprising administering to an individual inneed thereof an effective amount of an immunomodulatory composition ofthe present disclosure in a vaccine including an antigen that willmodulate the dysfunctional immune response to a disease related antigen.

Methods of Treating Neurological Disorders

The present disclosure provides methods of modulating and/or regulatingan inflammatory response, the method comprising administering to anindividual in need thereof an effective amount of an immunomodulatorycomposition of the present disclosure. Inflammatory conditions accountfor many neurological disorders such as Alzheimer's, schizophrenia,multiple sclerosis, Parkinson's disease, autism, Amyotrophic LateralSclerosis (ALS), Cerebral malaria disorders etc. By modulating innateand adaptive immune mechanisms through the immunomodulatory compositionof the present disclosure, neurological disorders can be treated.

In some cases, a method of the present disclosure of treating aneurological disorder comprises administering an immunomodulatorycomposition to an individual in need thereof, and further comprisingadministering to the individual an effective amount of at least oneadditional therapeutic agent.

In some cases, the method comprising administering to an individual inneed thereof an effective amount of an immunomodulatory composition ofthe present disclosure in a vaccine including an antigen that willelicit an immune response to a disease related protein such as theamyloid plaques characteristics of Alzheimer or Creutzfeldt-Jacobdisease (CJD).

Methods of Preventing or Treating Immunosuppression and InfectionsFollowing Stroke and Other Traumatic Brain Injuries

The present disclosure provides methods of preventing or limitinginfections following strokes and other brain injuries comprisingadministering an immunomodulatory composition of the present disclosureto an individual in need thereof. Various forms of brain trauma,including stroke, lead to long-term systemic immune suppression,resulting in higher infection and mortality rates. Further, hepaticinvarant NKT cells have been shown to be important to ameliorartesystemic immunosuppression. The present disclosure represents a strategyto prevent systemic immunosuppression and infections in these patientsthrough activation of NK, NKT and other immune cells.

In some cases, a method of the present disclosure of treating a strokeor brain trauma disorder comprises administering an immunomodulatorycomposition to an individual in need thereof, and further comprisingadministering to the individual an effective amount of at least oneadditional therapeutic agent.

Methods of Treating Addiction with Addictive Substances

The present disclosure provides methods of inducing antibody responsesagainst addictive substances such as such as nicotine, cocaine, heroin,etc. The methods generally involve administering to an individual inneed thereof an effective amount of an immunomodulatory composition ofthe present disclosure. In some cases, a method of the presentdisclosure of inducing an immune response to an addictive substancecomprises administering to an individual in need thereof an effectiveamount of an immunomodulatory composition of the present disclosure,where the immunomodulatory composition comprises the addictive substance(e.g., nicotine, cocaine, heroin, etc.). The intent is to immunizepatients with the vaccine comprising of the immunomodulatory compositionof the disclosure as a part of the vaccination; if the patient usescocaine after vaccination, the antibody will inhibit the reinforcingactivity of cocaine and decrease the likelihood of continued addiction.

In some cases, a method of the present disclosure of treating anaddiction comprises administering an immunomodulatory composition to anindividual in need thereof, and further comprising administering to theindividual an effective amount of at least one additional therapeuticagent.

Methods of Enhancing the Effectiveness of an Existing Vaccine

The present disclosure provides methods of enhancing and/or regulatingan immune response to an existing vaccine, the method comprisingadministering to an individual in need thereof an effective amount of animmunomodulatory composition of the present disclosure. In some cases, amethod of the present disclosure may allow reducing the dose of avaccine, modulating the route and scheduling of a vaccine, andincreasing protection in individuals with impaired immune responses. Insome cases, a method of the present disclosure of enhancing and/orregulating an immune response to a vaccine comprises administering to anindividual in need thereof an effective amount of an immunomodulatorycomposition of the present disclosure, where the immunomodulatorycomposition comprises an existing vaccine. Some non-limiting suitablevaccines are BCG, HBV vaccine Fenderix, Hepatitis A vaccine, Influenzavaccines (trivalent and tetravalent vaccines, Flumist, Nasovac),Rotavirus vaccine (Rota Teq, Rotarix), polio vaccine (trivalent,bivalent, monovalent vaccines), Diphtheria-tetanus vaccine, S. typhi(Vivotif, Ty21A), S. pneumoneae vaccine, E. coli vaccine, Pertussisvaccine, HPV vaccine Gardisil, measles vaccine, MMR vaccine,Meningococcal vaccine, Vibrio cholerae (Orochol), cholera (Dukoral) andother known vaccines.

Methods of Enhancing the Efficacy and/or Reducing the Toxicity of aTherapeutic Treatment

The present disclosure also provides methods for enhancing the efficacyand/or reducing the toxicity of a therapeutic treatment, preferablytreatment with an anti-infective or antiviral drug, anticancer, otherimmunostimulatory/modulatory compounds or a surgical treatment byadministering an effective amount of an immunomodulatory composition ofthe present disclosure to an individual, cells or tissues preferably theamount needed to elicit and/or regulate an immune response.

In some cases, a method of the present disclosure of enhancing efficacyand reducing toxicity comprises administering an immunomodulatorycomposition to an individual in need thereof, and further comprisingadministering to the individual an effective amount of at least oneadditional therapeutic agent.

Methods of Increasing Antigen Presentation on Dendritic Cells

The present disclosure provides a method of enhancing antigenpresentation on a dendritic cell, the method comprising: a) contactingdendritic cells (DCs) obtained from an individual with a compositioncomprising: i) heat-killed Caulobacter crescentus; and ii) an antigen.The DCs are contacted with the HKCC and the antigen is in vitro.Contacting DCs with the antigen and the HKCC enhances antigenpresentation of the antigen on the DCs, thereby generating a populationof antigen-presenting DCs. In some cases, the antigen can be contactedwith DCs using methods such as diffusion, electroporation, activetransport, liposome fusion, phagocytosis, sonication etc. In some cases,the method further comprises administering the antigen-presenting DCs tothe individual from whom the DCs were obtained. In some cases, themethod further comprises administering the antigen-presenting DCscombined with antibodies, chemotherapeutic agents, or cytokines to theindividual from whom the DCs were obtained. Administering anantigen-presenting DC to an individual can treat a disease in theindividual.

Suitable antigens are described above. In some cases, a compositioncomprising HKCC and antigen is contacted with DCs; and theHKCC-antigen-DC mixture is incubated for a period of time of from about30 minutes to about 48 hours, thereby generating a population ofantigen-presenting DCs. A subject method can increase the proportion ofDCs that are antigen-presenting DCs by at least about 25%, at leastabout 50%, at least about 75%, at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 25-fold, at least about50-fold, at least about 100-fold, or more than 100-fold, compared to theproportion of DCs in the starting population that are antigen-presentingDCs.

Methods of Activating Effector Immune Cells

The present disclosure provides a method of activating effectorlymphocytes such as NK, NKT, T cells, and B cells, the methodcomprising: a) contacting effector cells (NK, NKT, T cells, and/or Bcells) obtained from an individual with a composition comprising: i)heat-killed Caulobacter crescentus; and/or ii) an antigen in thepresence or absence of antigen presenting cells. Contacting effectorlymphocytes and the HKCC enhances their activation, thereby generating apopulation of activated effector lymphocytes. In some cases, naïve Tcells can be primed in vitro/ex vivo against a given antigen, comprisingcontacting naïve T cells with professional antigen presenting cells, anantigen and immunomodulatory composition of the present disclosure undersuitable conditions and sufficient time to activate the naïve T cells.In some cases, the method further comprises administering the activatedeffector lymphocytes to the individual from whom the cells wereobtained, to prevent and/or treat a disease in a host. In some cases,the method further comprises administering the activated effectorlymphocytes combined with antibodies, chemotherapeutic agents, orcytokines to the individual from whom the cells were obtained, toprevent and/or treat a disease in a host.

Methods of Treating an Infection with an Intracellular Pathogen

The present disclosure provides methods of preventing and/or treatinginfections with intracellular pathogens (e.g., viruses, mycobacteria,bacteria, parasites etc.) in an individual, the method comprisingadministering to the individual an effective amount of animmunomodulatory composition of the present disclosure.

In some cases, a method of the present disclosure of treating anintracellular pathogen comprises administering to an individual in needthereof, and further comprising administering to the individual aneffective amount of at least one additional therapeutic agent.

Methods of Enhancing Immune Responses in Cell Culture for Research,Diagnosis and/or Therapeutic Purposes

The present disclosure provides a method of activating various TLRs,NLRs, DCs and/or effector lymphocytes such as NK, NKT, T and B cells,the method comprising: a) contacting effector cells (NK, NKT, T and Bcells) obtained from an individual with a composition comprising: i)heat-killed Caulobacter crescentus; and/or ii) an antigen in thepresence or absence of antigen presenting cells. Contacting effectorlymphocytes and the HKCC enhances their activation, thereby generating apopulation of activated effector lymphocytes. In some cases, the methodcomprises of diagnosing a disease state by identifying and expandingspecific antigen reactive T cells and/or B cells. In some cases, themethod comprises of identifying and expanding specific antigen reactiveT cells and/or B cells in vitro for research purposes. In some cases themethod comprises of administering the activated effector lymphocytes tothe individual from whom the cells were obtained, to prevent and/ortreat a disease in a host. In some cases, the method comprises ofactivating TLRs or NLRs for research and/or diagnostic purposes.

Methods of Inducing Proliferation and Differentiation of Stem Cells

The present disclosure provides a method of inducing proliferation,differentiation of stem cells and restoration of homeostasis in anindividual, the method comprising administering to the individual aneffective amount of an immunomodulatory composition of the presentdisclosure. The present disclosure provides a method of modifying stemcells, the method comprising contacting the stem cells with acomposition comprising heat-killed Caulobacter crescentus, wherein saidcontacting generates a population of expanded and/or differentiated stemcells.

The present disclosure also provides a method of inducing proliferationand/or differentiation of stem cells, the method comprising contactingstem cells obtained from an individual with an immunomodulatorycomposition of the present disclosure, e.g., an immunomodulatorycomposition comprising heat-killed Caulobacter crescentus. Contactingthe stem cells with the HKCC leads to their proliferation anddifferentiation, thereby generating a population of expanded anddifferentiated cells. The population of expanded and differentiatedcells can then be administered to the individual from whom the stemcells were obtained.

In some embodiments, a method of the present disclosure of inducingproliferation and/or differentiation of stem cells comprises: a)obtaining stem cells from an individual; b) contacting the stem cells invitro with HKCC, thereby generating a population of expanded anddifferentiated cells; and c) administering the population of expandedand differentiated cells to the individual.

In some embodiments, a method of the present disclosure of inducingproliferation and/or differentiation of stem cells in an individualcomprises administering to the individual an effective amount of animmunomodulatory composition of the present disclosure. In some cases,an effective amount of an immunomodulatory composition of the presentdisclosure is an amount that is effective, when administered in a singledose or in multiple doses, to induce proliferation and/ordifferentiation of hematpoietic stem cells, and restore homeostasis. Forexample, in some cases, an effective amount of an immunomodulatorycomposition of the present disclosure is an amount that is effective,when administered in a single dose or in multiple doses, to induceproliferation and/or differentiation of hematpoietic stem cells, andrestore homeostasis in an individual by at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 75%, at least 100% (or 2-fold), atleast 2.5-fold, at least 5-fold, at least 10-fold, more than 10-fold, atleast 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, atleast 100-fold, or more than 100-fold, compared to the individual in theabsence of treatment with the immunomodulatory composition.

Formulations, Dosages, and Routes of Administration

An immunomodulatory composition of the present disclosure can includeone or more pharmaceutically acceptable excipients; and can beformulated in any of a variety of ways, that may depend, e.g., on theroute of administration. Pharmaceutically acceptable excipients areknown to those skilled in the art, and have been amply described in avariety of publications, including, for example, A. Gennaro (1995)“Remington: The Science and Practice of Pharmacy”, 19th edition,Lippincott, Williams, & Wilkins. Suitable excipient vehicles include,for example, water, saline, dextrose, glycerol, ethanol, inert proteins,hydrophillic polymers, amino acids, fatty acids, surfactants, non-ionicsurfactants, carbohydrates, dextrins, polyols, chelating agents, or thelike, and combinations thereof. In addition, if desired, the vehicle maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents or pH buffering agents. Actual methods of preparingsuch dosage forms are known, or will be apparent, to those skilled inthe art. See, e.g., Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., 17th edition, 1985; Remington: The Science andPractice of Pharmacy, A. R. Gennaro, (2000) Lippincott, Williams &Wilkins.

An immunomodulatory composition can be incorporated into a variety offormulations for therapeutic administration. More particularly, animmunomodulatory composition can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers, salts, preservatives, buffering agents, ordiluents, and may be formulated into preparations in solid, semi-solid,liquid, lyophilized or gaseous forms, such as tablets, capsules,powders, granules, ointments, solutions, suppositories, injections, skinpatches, inhalants and aerosols. In other embodiments, the formulationcomprises a colloidal delivery system that includes e.g., liposomes,nano-particles, nano-emulsions, nano capsules, microspheres andpolymers.

In pharmaceutical dosage forms, an immunomodulatory composition may beadministered alone or in appropriate association, as well as incombination, with other pharmaceutically active compounds. Animmunomodulatory composition, an antigen, adjuvant and/or therapeuticdrug can be administered concurrently, simultaneously, sequentially orat different times, at the same or different sites, and via differentroutes. The following methods and excipients are merely exemplary andare in no way limiting.

For oral preparations, an immunomodulatory composition can be used aloneor in combination with appropriate additives to make tablets, powders,granules or capsules, for example, with conventional additives, such aslactose, mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

An immunomodulatory composition can be formulated into liquidpreparations for administration by dissolving, suspending or emulsifyingthe composition in an aqueous or nonaqueous solvent, such as vegetableor other similar oils, synthetic aliphatic acid glycerides, esters ofhigher aliphatic acids or propylene glycol; and if desired, withconventional additives such as solubilizers, isotonic agents, suspendingagents, emulsifying agents, stabilizers and preservatives.

An immunomodulatory composition can be utilized in aerosol formulationto be administered via inhalation. The immunomodulatory compositions ofthe present disclosure can be formulated into pressurized acceptablepropellants such as dichlorodifluoromethane, propane, nitrogen and thelike.

Furthermore, an immunomodulatory composition can be made intosuppositories by mixing with a variety of bases such as emulsifyingbases or water-soluble bases. An immunomodulatory composition can beadministered rectally via a suppository. The suppository can includevehicles such as cocoa butter, carbowaxes and polyethylene glycols,which melt at body temperature, yet are solidified at room temperature.

An immunomodulatory composition of the present disclosure can also beadministered in the form of liposomes or liposomal polymeric gels.Liposomes can be given by a variety of routes, oral, nasal, parenteral,trans-dermal, inhalation etc. As is known in the art, liposomes arederived from phospholipids or other lipid substances. Liposomes areformed by mono- or multilamellar hydrated liquid crystals that aredispersed in an aqueous medium. Any non-toxic, physiologicallyacceptable and metabolizable lipid capable of forming liposomes can beused. The present compositions in liposome form can contain, in additionto an immunomodulatory composition of the present disclosure, one ormore of a stabilizer, a preservative, an excipients, and the like.Exemplary lipids are the phospholipids and the phosphatidylcholines(lecithins), both natural and synthetic. Liposomes can be in a sizerange of from less than 100 nm to several microns. Methods to formliposomes are known in the art. for example, Prescott, Ed., Methods inCell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33et seq.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, emulsions, and suspensions may be provided wherein each dosageunit, for example, teaspoonful, tablespoonful, tablet or suppository,contains a predetermined amount of the composition containing one ormore active agents. Similarly, unit dosage forms for injection orintravenous administration may comprise an immunomodulatory compositionas a solution in sterile water, normal saline or anotherpharmaceutically acceptable carrier.

A subject immunomodulatory composition can be formulated for topicaladministration. Topical administration includes administration to theskin or mucosa, including surfaces of the lung eye, nose, and ear.Suitable topical preparations include, e.g., skin patch preparation,transdermal patch preparation, micro arrays, cream, lotion, gelpreparations, powder, ointment, paste, intranasal drops or gels.

Ointments are semi-solid preparations, which are typically based onpetrolatum or other petroleum derivatives. Suitable ointments includeoleaginous bases; emulsifiable bases; emulsion bases; and water-solublebases. Oleaginous ointment bases include, for example, vegetable oils,fats obtained from animals, and semisolid hydrocarbons obtained frompetroleum. Emulsifiable ointment bases, also known as absorbent ointmentbases, contain little or no water and include, for example,hydroxystearin sulfate, anhydrous lanolin and hydrophilic petrolatum.Emulsion ointment bases are either water-in-oil (WIO) emulsions oroil-in-water (OIW) emulsions, and include, for example, cetyl alcohol,glyceryl monostearate, lanolin and stearic acid. Exemplary water-solubleointment bases are prepared from polyethylene glycols of varyingmolecular weight.

Lotions are preparations to be applied to the skin surface withoutfriction, and are typically liquid or semi liquid preparations in whichsolid particles, including the active agent, are present in a water oralcohol base. Lotions are usually suspensions of solids, and preferably,for the present purpose, comprise a liquid oily emulsion of theoil-in-water type. Lotions can be used for treating large body areas,because of the ease of applying a more fluid composition. Lotions maycontain suspending agents to produce better dispersions as well ascompounds useful for localizing and holding the active agent in contactwith the skin, e.g., methyl cellulose, sodium carboxymethyl-cellulose,or the like. An example of a lotion formulation for use in conjunctionwith the present invention contains propylene glycol mixed with ahydrophilic petrolatum such as that which may be obtained under thetrademark Aquaphor® from Beiersdorf, Inc. (Norwalk, Coon.).

Suitable creams can be viscous liquid or semisolid emulsions, eitheroil-in-water or water-in-oil. Cream bases are water-washable, andcontain an oil phase, an emulsifier and an aqueous phase. The oil phase,also sometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil sophase in volume, and generally contains a humectant. The emulsifier in acream formulation, as explained in Remington, supra, is generally anonionic, anionic, cationic or amphoteric surfactant.

Gels formulations can be used. Gels are semisolid, suspension-/typesystems. Single-phase gels contain organic macromolecules distributedsubstantially uniformly throughout the carrier liquid, which can beaqueous, but may also contain an alcohol and, optionally, an oil.

A topical formulation may also be delivered to the skin usingconventional “transdermal”-type patches, wherein the agent(immunomodulatory composition) is contained within a laminated structurethat serves as a delivery device to be affixed to the skin. In such astructure, the immunomodulatory composition is contained in a layer, or“reservoir,” underlying an upper backing layer. The laminated structuremay contain a single reservoir, or it may contain multiple reservoirs.In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysioxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. The particular polymeric adhesive selectedwill depend on the particular immunomodulatory composition, vehicle,etc., i.e., the adhesive must be compatible with all components of thedrug-containing composition. In an alternative embodiment, theimmunomodulatory composition-containing reservoir and skin contactadhesive are present as separate and distinct layers, with the adhesiveunderlying the reservoir which, in this case, may be either a polymericmatrix as described above, or it may be a liquid or hydrogel reservoir,or may take some other form.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of an activeagent (e.g., HKCC; antigen; etc.) calculated in an amount sufficient toproduce the desired effect in association with a pharmaceuticallyacceptable diluent, carrier or vehicle. The specifications for theactive agents depend on the particular compound employed and the effectto be achieved, and the pharmacodynamics associated with each compoundin the host.

Other modes of administration will also find use. For instance, animmunomodulatory composition can be formulated in suppositories and, insome cases, aerosol and intranasal compositions. For suppositories, thevehicle composition will include traditional binders and carriers suchas, polyalkylene glycols, or triglycerides. Such suppositories may beformed from mixtures containing the active ingredient in the range ofabout 0.5% to about 10% (w/w), or about 1% to about 2%.

Intranasal formulations will usually include vehicles that neither causeirritation to the nasal mucosa nor significantly disturb ciliaryfunction. Diluents such as water, aqueous saline or other knownsubstances can be employed. The nasal formulations may also containpreservatives such as, but not limited to, chlorobutanol andbenzalkonium chloride. A surfactant may be present to enhance absorptionof the subject proteins by the nasal mucosa.

An immunomodulatory composition can be administered as injectables.Typically, injectable compositions are prepared as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared. The preparationmay also be emulsified or the active ingredient encapsulated in liposomevehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985; Remington: The Science and Practice of Pharmacy, A. R.Gennaro, (2000) Lippincott, Williams & Wilkins. The composition orformulation to be administered will, in any event, contain a quantity ofan active agent (e.g., HKCC; antigen; etc.) adequate to achieve thedesired state in the subject being treated.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,salts, carriers or diluents, are readily available to the public.Moreover, pharmaceutically acceptable auxiliary substances, such as pHadjusting and buffering agents, tonicity adjusting agents, stabilizers,emulsifying agents, surfactants, preservatives, amino acids, fattyacids, wetting agents and the like, are readily available to the public.

Oral Formulations

In some embodiments, an immunomodulatory composition is formulated fororal delivery to an individual in need of such an immunomodulatorycomposition.

For oral delivery, a subject formulation comprising an immunomodulatorycomposition will in some embodiments include an enteric-soluble coatingmaterial. Suitable enteric-soluble coating material includehydroxypropyl methylcellulose acetate succinate (HPMCAS), hydroxypropylmethyl cellulose phthalate (HPMCP), cellulose acetate phthalate (CAP),polyvinyl phthalic acetate (PVPA), Eudragit™, and shellac.

Suitable oral formulations also include an immunomodulatory composition,formulated with any of the following: microgranules (see, e.g., U.S.Pat. No. 6,458,398); biodegradable macromers (see, e.g., U.S. Pat. No.6,703,037); biodegradable hydrogels (see, e.g., Graham and McNeill(1989) Biomaterials 5:27-36); biodegradable particulate vectors (see,e.g., U.S. Pat. No. 5,736,371); bioabsorbable lactone polymers (see,e.g., U.S. Pat. No. 5,631,015); slow release protein polymers (see,e.g., U.S. Pat. No. 6,699,504; Pelias Technologies, Inc.); apoly(lactide-co-glycolide/polyethylene glycol block copolymer (see,e.g., U.S. Pat. No. 6,630,155; Atrix Laboratories, Inc.); a compositioncomprising a biocompatible polymer and particles of metalcation-stabilized agent dispersed within the polymer (see, e.g., U.S.Pat. No. 6,379,701; Alkermes Controlled Therapeutics, Inc.); andmicrospheres (see, e.g., U.S. Pat. No. 6,303,148; Octoplus, B.V.).

Suitable oral formulations also include an immunomodulatory compositionformulated with any of the following: a carrier such as Emisphere®(Emisphere Technologies, Inc.); TIMERx, a hydrophilic matrix combiningxanthan and locust bean gums which, in the presence of dextrose, form astrong binder gel in water (Penwest); Geminex™ (Penwest); Procise™(GlaxoSmithKline); SAVIT™ (Mistral Pharma Inc.); RingCap™ (Alza Corp.);Smartrix® (Smartrix Technologies, Inc.); SQZgel™ (MacroMed, Inc.);Geomatrix™ (Skye Pharma, Inc.); Oros® Tri-layer (Alza Corporation); andthe like.

Also suitable for use are formulations such as those described in U.S.Pat. No. 6,296,842 (Alkermes Controlled Therapeutics, Inc.); U.S. Pat.No. 6,187,330 (Scion, Inc.); and the like.

Also suitable for use herein are formulations comprising an intestinalabsorption enhancing agent. Suitable intestinal absorption enhancersinclude, but are not limited to, calcium chelators (e.g., citrate,ethylenediamine tetracetic acid); surfactants (e.g., sodium dodecylsulfate, bile salts, palmitoylcarnitine, and sodium salts of fattyacids); toxins (e.g., zonula occludens toxin); and the like.

Suitable oral formulations also include an immunomodulatory composition,formulated as a food supplement (e.g. nutraceuticals, yogurt, bars,drinks, prebiotics, symbiotics, paraprobiotics) etc.

Controlled Release Formulations

In some embodiments, an immunomodulatory composition is formulated in acontrolled release formulation.

Controlled release can be taken to mean any one of a number of extendedrelease dosage forms. The following terms may be considered to besubstantially equivalent to controlled release, for the purposes of thepresent invention: continuous release, controlled release, delayedrelease, depot, gradual release, long-term release, programmed release,prolonged release, proportionate release, protracted release,repository, retard, slow release, spaced release, sustained release,time coat, timed release, delayed action, extended action, layered-timeaction, long acting, prolonged action, repeated action, slowing acting,sustained action, sustained-action medications, and extended release.Further discussions of these terms may be found in Lesczek Krowczynski,Extended-Release Dosage Forms, 1987 (CRC Press, Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems.

Physical systems include, but are not limited to, reservoir systems withrate-controlling membranes, such as microencapsulation,macroencapsulation, and membrane systems; reservoir systems withoutrate-controlling membranes, such as hollow fibers, ultra microporouscellulose triacetate, and porous polymeric substrates and foams;monolithic systems, including those systems physically dissolved innon-porous, polymeric, or elastomeric matrices (e.g., nonerodible,erodible, environmental agent ingression, and degradable), and materialsphysically dispersed in non-porous, polymeric, or elastomeric matrices(e.g., nonerodible, erodible, environmental agent ingression, anddegradable); laminated structures, including reservoir layers chemicallysimilar or dissimilar to outer control layers; and other physicalmethods, such as osmotic pumps, or adsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release may be found in Agis F. Kydonieus, Controlled ReleaseTechnologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

There are a number of controlled release drug formulations that aredeveloped for oral administration. These include, but are not limitedto, osmotic pressure-controlled gastrointestinal delivery systems;hydrodynamic pressure-controlled gastrointestinal delivery systems;

membrane permeation-controlled gastrointestinal delivery systems, whichinclude microporous membrane permeation-controlled gastrointestinaldelivery devices; gastric fluid-resistant intestine targetedcontrolled-release gastrointestinal delivery devices; geldiffusion-controlled gastrointestinal delivery systems; andion-exchange-controlled gastrointestinal delivery systems, which includecationic and anionic drugs. Additional information regarding controlledrelease drug delivery systems may be found in Yie W. Chien, Novel DrugDelivery Systems, 1992 (Marcel Dekker, Inc.). Some of these formulationswill now be discussed in more detail.

Enteric coatings are applied to tablets to prevent the release of activeagents in the stomach either to reduce the risk of unpleasant sideeffects or to maintain the stability of the drug which might otherwisebe subject to degradation of expose to the gastric environment. Mostpolymers that are used for this purpose are polyacids that function byvirtue or the fact that their solubility in aqueous medium ispH-dependent, and they require conditions with a pH higher than normallyencountered in the stomach.

One exemplary type of oral controlled release structure is entericcoating of a solid or liquid dosage form. The enteric coatings aredesigned to disintegrate in intestinal fluid for ready absorption. Delayof absorption of the active agent that is incorporated into aformulation with an enteric coating is dependent on the rate of transferthrough the gastrointestinal tract, and so the rate of gastric emptyingis an important factor. Some investigators have reported that amultiple-unit type dosage form, such as granules, may be superior to asingle-unit type.

Suitable enteric coating agents include, but are not limited to,hydroxypropylmethylcellulose phthalate, methacryclic acid-methacrylicacid ester copolymer, polyvinyl acetate-phthalate and cellulose acetatephthalate.

Another type of useful oral controlled release structure is a soliddispersion. A solid dispersion may be defined as a dispersion of one ormore active ingredients in an inert carrier or matrix in the solid stateprepared by the melting (fusion), solvent, or melting-solvent method.

Examples of carriers useful in solid dispersions include, but are notlimited to, water-soluble polymers such as polyethylene glycol,polyvinylpyraolidone, and hydroxypropylmethylcellulose. Alternativecarriers include phosphatidylcholine. Phosphatidylcholine is anamphoteric but water-insoluble lipid, which may improve the solubilityof otherwise insoluble active agents in an amorphous state inphosphatidylcholine solid dispersions.

Other carriers include polyoxyethylene hydrogenated castor oil. Animmunomodulatory composition can be included in a solid dispersionsystem with an enteric polymer such as hydroxypropylmethylcellulosephthalate and carboxymethylethylcellulose, and a non-enteric polymer,hydroxypropylmethylcellulose. Another solid dispersion dosage formincludes incorporation of the drug of interest (e.g., an active agent)with ethyl cellulose and stearic acid in different ratios.

There are various methods commonly known for preparing soliddispersions. These include, but are not limited to, the melting method,the solvent method and the melting-solvent method.

Injectable microspheres are another controlled release dosage form.Injectable micro spheres may be prepared by non-aqueous phase separationtechniques, and spray-drying techniques. Microspheres may be preparedusing polylactic acid or copoly(lactic/glycolic acid).

Other controlled release technologies that may be used include, but arenot limited to, SODAS (Spheroidal Oral Drug Absorption System), INDAS(Insoluble Drug Absorption System), IPDAS (Intestinal Protective DrugAbsorption System), MODAS (Multiporous Oral Drug Absorption System),EFVAS (Effervescent Drug Absorption System), PRODAS (Programmable OralDrug Absorption System), and DUREDAS (Dual Release Drug AbsorptionSystem) available from Elan Pharmaceutical Technologies. SODAS are multiparticulate dosage forms utilizing controlled release beads. INDAS are afamily of drug delivery technologies designed to increase the solubilityof poorly soluble drugs. IPDAS are multi particulate tablet formationutilizing a combination of high density controlled release beads and animmediate release granulate. MODAS are controlled release single unitdosage forms. Each tablet consists of an inner core surrounded by asemipermeable multiparous membrane that controls the rate of drugrelease. EFVAS is an effervescent drug absorption system. PRODAS is afamily of multi particulate formulations utilizing combinations ofimmediate release and controlled release mini-tablets. DUREDAS is abilayer tablet formulation providing dual release rates within the onedosage form. Although these dosage forms are known to one of skill,certain of these dosage forms will now be discussed in more detail.

An immunomodulatory composition of the present disclosure can beincorporated into any one of the aforementioned controlled releaseddosage forms, or other conventional dosage forms. The amount of activeagent contained in each dose can be adjusted, to meet the needs of theindividual patient, and the indication. One of skill in the art andreading this disclosure will readily recognize how to adjust the levelof an active agent and the release rates in a controlled releaseformulation, in order to optimize delivery of an active agent and itsbioavailability.

Inhalational Formulations

An immunomodulatory composition of the present disclosure will in someembodiments be administered to a patient by means of a pharmaceuticaldelivery system for the inhalation route. The immunomodulatorycomposition may be formulated in a form suitable for administration byinhalation. The inhalational route of administration provides theadvantage that the inhaled drug can bypass the blood-brain barrier. Thepharmaceutical delivery system is one that is suitable for respiratorytherapy by delivery of an active agent to mucosal linings of thebronchi. A system that depends on the power of a compressed gas to expelthe immunomodulatory composition from a container can also be used. Anaerosol or pressurized package can be employed for this purpose.

As used herein, the term “aerosol” is used in its conventional sense asreferring to very fine liquid or solid particles carries by a propellantgas under pressure to a site of therapeutic application. When apharmaceutical aerosol is employed, the aerosol contains thetherapeutically active compound (e.g., active agent), which can bedissolved, suspended, or emulsified in a mixture of a fluid carrier anda propellant. The aerosol can be in the form of a solution, suspension,emulsion, powder, or semi-solid preparation. Aerosols can be used foradministration as fine, solid particles or as liquid mists via therespiratory tract of a patient. Various types of propellants known toone of skill in the art can be utilized. Suitable propellants include,but are not limited to, hydrocarbons or other suitable gas. In the caseof the pressurized aerosol, the dosage unit may be determined byproviding a value to deliver a metered amount.

An immunomodulatory composition can also be formulated for delivery witha nebulizer, which is an instrument that generates very fine liquidparticles of substantially uniform size in a gas. For example, a liquidcontaining the immunomodulatory composition is dispersed as droplets.The small droplets can be carried by a current of air through an outlettube of the nebulizer. The resulting mist penetrates into therespiratory tract of the patient.

There are several different types of inhalation methodologies which canbe employed in connection with an immunomodulatory composition of thepresent disclosure. An immunomodulatory composition can be formulatedwith low boiling point propellants. Such formulations are generallyadministered by conventional meter dose inhalers (MDI's). Alternatively,immunomodulatory composition can be formulated in aqueous or ethanolicsolutions and delivered by conventional nebulizers. In some embodiments,such solution formulations are aerosolized using devices and systemssuch as disclosed within U.S. Pat. Nos. 5,497,763; 5,544,646; 5,718,222;and 5,660,166. An immunomodulatory composition can be formulated intodry powder formulations. Such formulations can be administered by simplyinhaling the dry powder formulation after creating an aerosol mist ofthe powder. Technology for carrying such out is described within U.S.Pat. No. 5,775,320 issued Jul. 7, 1998 and U.S. Pat. No. 5,740,794issued Apr. 21, 1998.

An immunomodulatory composition of the present disclosure will in someembodiments be formulated for vaginal delivery. A subjectimmunomodulatory composition for intravaginal administration can beformulated as an intravaginal bioadhesive tablet, intravaginalbioadhesive microparticle, intravaginal cream, intravaginal lotion,intravaginal foam, intravaginal ointment, intravaginal paste,intravaginal solution, or intravaginal gel.

A subject immunomodulatory composition will in some embodiments beformulated for rectal delivery. A subject formulation for intrarectaladministration comprises a subject immunomodulatory compositionformulated as an intrarectal bioadhesive tablet, intrarectal bioadhesivemicroparticle, intrarectal cream, intrarectal lotion, intrarectal foam,intrarectal ointment, intrarectal paste, intrarectal solution, orintrarectal gel. An immunomodulatory composition of the presentdisclosure can be formulated with agents that improve adhesion tomucosal membranes such as mucoadhesives, bioadhesives, particles,microspheres or liposomes.

A subject immunomodulatory composition can include one or more of anexcipient (e.g., sucrose, starch, mannitol, sorbitol, lactose, glucose,cellulose, talc, calcium phosphate or calcium carbonate), a binder(e.g., cellulose, methylcellulose, hydroxymethylcellulose,polypropylpyrrolidone, polyvinylpyrrolidone, gelatin, gum arabic,poly(ethylene glycol), sucrose or starch), a disintegrator (e.g.,starch, carboxymethylcellulose, hydroxypropyl starch, low substitutedhydroxypropylcellulose, sodium bicarbonate, calcium phosphate or calciumcitrate), a lubricant (e.g., magnesium stearate, light anhydrous silicicacid, talc or sodium lauryl sulfate), a flavoring agent (e.g., citricacid, menthol, glycine or orange powder), a preservative (e.g., sodiumbenzoate, sodium bisulfite, methylparaben or propylparaben), astabilizer (e.g., citric acid, sodium citrate or acetic acid), asuspending agent (e.g., methylcellulose, polyvinylpyrrolidone oraluminum stearate), a dispersing agent (e.g.,hydroxypropylmethylcellulose), a diluent (e.g., water), and base wax(e.g., cocoa butter, white petrolatum or polyethylene glycol).

Tablets comprising an immunomodulatory composition may be coated with asuitable film-forming agent, e.g., hydroxypropylmethyl cellulose,hydroxypropyl cellulose or ethyl cellulose, to which a suitableexcipient may optionally be added, e.g., a softener such as glycerol,propylene glycol, diethylphthalate, or glycerol triacetate; a fillersuch as sucrose, sorbitol, xylitol, glucose, or lactose; a colorant suchas titanium hydroxide; and the like.

Dosages

The dosage of an immunomodulatory composition of the present disclosurecan vary, depending on factors such as the clinical goals to beachieved, the age of the individual being treated, the physical statusof the individual being treated, etc.

An immunomodulatory composition of the present disclosure can compriseHKCC in an amount of from about 10³ HKCC per unit dosage form to about10²⁰ HKCC per unit dosage form. For example, an immunomodulatorycomposition of the present disclosure can comprise HKCC in an amount offrom about 10³ HKCC per unit dosage form to about 10⁴ HKCC per unitdosage form, from about 10⁴ HKCC per unit dosage form to about 10⁵ HKCCper unit dosage form, from about 10⁵ HKCC per unit dosage form to about10⁶ HKCC per unit dosage form, from about 10⁶ HKCC per unit dosage formto about 10⁷ HKCC per ml, from about 10⁸ HKCC per unit dosage form toabout 10⁹ HKCC per unit dosage form, from about 10⁹ HKCC per ml to about10¹⁰ HKCC per unit dosage form, from about 10¹⁵ HKCC per unit dosageform to about 10²⁰ HKCC per unit dosage form, or more than 10²⁰ HKCC perunit dosage form.

For example, an immunomodulatory composition of the present disclosurecan comprise HKCC in an amount of from about 10³ HKCC per nil to about10²⁰ HKCC per ml. For example, an immunomodulatory composition of thepresent disclosure can comprise HKCC in an amount of from about 10³ HKCCper ml to about 10⁴ HKCC per ml, from about 10⁴ HKCC per ml to about 10⁵HKCC per ml, from about 10⁵ HKCC per ml to about 10⁶ HKCC per ml, fromabout 10⁶ HKCC per ml to about 0⁷ HKCC per ml, from about 10⁸ HKCC perml to about 10⁹ HKCC per ml, from about 10⁹ HKCC per ml to about 10¹⁰HKCC per ml, from about 10¹⁵ HKCC per ml to about 10²⁰ HKCC per ml, ormore than 10²⁰ HKCC per ml.

An immunomodulatory composition of the present disclosure can compriseHKCC in an amount of from about 10² to about 10²⁰ colony forming units(cfu) per unit dosage form; for example, an immunomodulatory compositionof the present disclosure can comprise HKCC in an amount of from about10² to about 10³ from about 10³ to about 10⁵, from about 10⁵ to about10⁷, from about 10⁷ to about 10⁹, from about 10⁹ to about 10¹¹, fromabout 10¹¹ to about 10¹³, from about 10¹³ to about 10¹⁵, from about 10¹⁵to about 10¹⁸, or from about 10¹⁸ to about 10²⁰, cfu per unit dosageform. A unit dosage form can be an amount that is administered in asingle dose; for example, a unit dosage form can be 0.5 ml, 1.0 ml, orother volume suitable for administration in a single dose.

An immunomodulatory composition of the present disclosure can compriseHKCC in an amount of from about 10² to about 10²⁰ cfu per ml; forexample, an immunomodulatory composition of the present disclosure cancomprise HKCC in an amount of from about 10² to about 10³ from about 10³to about 10⁵, from about 10⁵ to about 10⁷, from about 10⁷ to about 10⁹,from about 10⁹ to about 10¹¹, from about 10¹¹ to about 10¹³, from about10¹³ to about 10¹⁵, from about 10¹⁵ to about 10¹⁸, or from about 10¹⁸ toabout 10²⁰, cfu per ml.

In some embodiments, multiple doses of an immunomodulatory compositionof the present disclosure are administered. The frequency ofadministration of an immunomodulatory composition of the presentdisclosure can vary depending on any of a variety of factors, e.g.,severity of the symptoms, etc. For example, in some embodiments, animmunomodulatory composition of the present disclosure is administeredonce per month, twice per month, three times per month, every other week(qow), once per week (qw), twice per week (biw), three times per week(tiw), four times per week, five times per week, six times per week,every other day (qod), daily (qd), twice a day (qid), or three times aday (tid).

The duration of administration of an immunomodulatory composition of thepresent disclosure, e.g., the period of time over which animmunomodulatory composition of the present disclosure is administered,can vary, depending on any of a variety of factors, e.g., patientresponse, etc. For example, an immunomodulatory composition of thepresent disclosure can be administered over a period of time rangingfrom about one hour to one day, from about one day to about one week,from about two weeks to about four weeks, from about one month to abouttwo months, from about two months to about four months, from about fourmonths to about six months, from about six months to about eight months,from about eight months to about 1 year, from about 1 year to about 2years, or from about 2 years to about 4 years, or more.

Where an immunomodulatory composition comprises an antigen, the dosageof antigen is selected as an amount which is effective and modulates animmune response without significant adverse side effects. Such amountcan vary, depending, e.g., upon which specific antigen is employed, theroute of administration, etc. Where an immunomodulatory compositioncomprises an antigen, the dosage of antigen can range from 1 ng per unitdosage form to about 100 mg per unit dosage form, e.g., from about 1 ngto about 25 ng, from about 25 ng to about 50 ng, from about 50 ng toabout 100 ng, from about 100 ng to about 250 ng, from about 250 ng toabout 500 ng, from about 500 ng to about 750 ng, from about 750 ng toabout 1 μg, from about 1 μg to about 25 μg, from about 25 μg to about 50μg, from about 50 μg to about 100 μg, from about 100 μg to about 250 μg,from about 250 μg to about 500 μg, from about 500 μg to about 750 μg,from about 750 μg to about 1 mg, from about 1 mg to about 25 mg, fromabout 25 mg to about 50 mg, or from about 50 mg to about 100 mg, perunit dosage form.

Routes of Administration

An immunomodulatory composition of the present disclosure isadministered to an individual using any available method and routesuitable for drug delivery, including in vivo and ex vivo methods, aswell as systemic and localized routes of administration.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, intranodal, percutaneous, transdermal, intratumoral,topical application, intravenous, intravesicular, rectal, nasal, oraland other enteral and parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe agent and/or the desired effect. The composition can be administeredin a single dose or in multiple doses.

An immunomodulatory composition of the present disclosure can beadministered to a host using any available conventional methods androutes suitable for delivery of conventional drugs, including systemicor localized routes. In general, routes of administration contemplatedinclude, but are not necessarily limited to, enteral, parenteral, orinhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intradermal, intralymphatic, intraorbital,intracapsular, intraspinal, intrasternal, intracranial, intravesicular,and intravenous routes, i.e., any route of administration other thanthrough the alimentary canal. Parenteral administration can be carriedto effect systemic or local delivery of the immunomodulatorycomposition. Where systemic delivery is desired, administrationtypically involves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

An immunomodulatory composition of the present disclosure can also bedelivered to the subject by enteral administration. Enteral routes ofadministration include, but are not necessarily limited to, oral andrectal (e.g., using a suppository) delivery.

An immunomodulatory composition of the present disclosure can also bedelivered to the subject via a mucosal route of delivery. Mucosal routesof delivery include nasal, buccal, sublingual, vaginal, ocular, andrectal routes of administration.

In certain embodiments, an immunomodulatory composition of the presentdisclosure is administered to a subject via a combination of differentroutes in the order indicated below:

i. systemic, mucosal;

ii. systemic, systemic, mucosal, mucosal;

iii. systemic, mucosal, systemic;

iv. mucosal, mucosal, systemic, systemic;

v. mucosal, systemic, systemic;

vi. mucosal, systemic, mucosal, for example.

When an immunomodulatory composition of the present disclosure isadministered systemically or mucosally more than once, the two or moresystemic or mucosal administrations may be by the same systemic (forexample, two intramuscular injections) or mucosal route (two IN/SLadministrations) or different (for example, one intramuscular injectionand one intravenous injection; one IN administration and one SLadministration).

An immunomodulatory composition of the present disclosure isadministered to an individual using any available method, delivery ordevice such as vaccine patches, needles, microneedles (hollow or solid),drop, syrup, tablets, capsules, pipette, dose-spray pumps, nasaldropper, inhalation devices, liquid or dry powder, suspensions orsolutions, spray devices, Accuspray™, thermoresponsive gels, jetinjectors, Nasovak™, Bespak™, ointment, lotions, suppositories, gelsetc.

Individuals Suitable for Treatment

Individuals suitable for treatment using a method of the presentdisclosure include humans; non-human mammals; fish; and birds. In any ofthe above embodiments discussed below, the individual being treatedusing a subject method can be a non-human mammal such as livestock(e.g., pigs, sheep, goats, tattles, equine, caprine, ovine, bovine,etc.); a mammalian pet (e.g., cats; dogs; horses; etc.); a bird such aschicken, hens, turkeys, geese, quail, ducks etc.; or other animals suchas fish.

In any of the above embodiments discussed below, the individual beingtreated using a subject method is a human of from about one month toabout 6 months, from about 6 months to about 1 year, or from about 1year to about 5 years of age. In any of the above embodiments discussedbelow, the individual being treated using a subject method is a human offrom about 5 years to about 12 years, from about 13 years to about 18years, or from about 18 years to about 25 years of age. In any of theabove embodiments discussed below, the individual being treated using asubject method is a human of from about 25 years to about 50 years, fromabout 50 years to about 75 years of age, or older than 75 years of age.In any of the above embodiments discussed below, the individual beingtreated using a subject method is a human who is immunocompromised.

In some embodiments, the individual has a viral disease, or is at riskof contracting a viral disease. In some cases, the disease is a viraldisease selected from the group consisting of, but not limited to, viraldisease caused by hepatitis B, hepatitis C, rotavirus, humanimmunodeficiency virus, human T-cell lymphotropic virus, DNA virusessuch as parvoviruses, adeno viruses, papovaviruses (e.g., papillomavirus, polyoma viruses, and SV40), herpes viruses (e.g., herpes simplextype I (HSV-I), herpes simplex type II (HSV-II), and Epstein-Barrvirus), poxviruses (e.g., variola (smallpox) and vaccinia virus); andRNA viruses, such as retroviruses [e.g. human immunodeficiency virustype I (HIV-I), human immunodeficiency virus type II (HIV-II), humanT-cell lymphotropic virus type I (HTLV-I), human T-cell lymphotropicvirus type II (HTLV-II)], orthomyxoviruses (e.g., influenza viruses),paramyxoviruses (e.g., measles virus, mumps virus, respiratory syncytialvirus), rhabdoviruses (e.g., rabies virus), Sendai virus, picornaviruses(e.g., poliomyelitis virus, coxsackieviruses, rhinoviruses), reoviruses(e.g., rotavirus, colorado tick fever virus), togaviruses (e.g., rubellavirus (German measles), Japanese encephalitis virus and Semliki forestvirus), arboviruses, calciviruses (e.g., hepatitis E virus),flaviviruses (e.g., yellow fever virus, dengue virus), coronaviruses,filoviruses (e.g., Ebola and Marburg viruses) and Bunyaviruses (e.g.,Hanta virus, California encephalitis virus).

In some embodiments, the individual has a bacterial infection, or is arisk of contracting a bacterial infection. In some embodiments, theindividual has a mycobacterial infection, or is at risk of contracting amycobacterial infection. In some embodiments, the individual is infectedwith, or is at risk of becoming infected with, a pathogenic bacterium.Pathogenic bacteria include, e.g., Gram positive bacteria, Gram negativebacteria, mycobacteria, etc. Non-limiting examples of pathogenicbacteria include Mycobacteria (e.g., M. tuberculosis, M. avium complex),Streptococcus, Staphylococcus, Pseudomonas, Salmonella, Neisseria, andListeria. In some cases, the bacteria is Neisseria gonorrhea, M.tuberculosis, M. leprae, Listeria monocytogenes, Streptococcuspneumoniae, S. pyogenes, S. agalactiae, S. viridans, S. faecalis, or S.bovis. Other examples of pathogenic bacteria contemplated include, butare not limited to, Gram positive bacteria (e.g., Listeria, Bacillussuch as Bacillus anthracis, Erysipelothrix species), Gram negativebacteria (e.g., Bartonella, Brucella, Campylobacter, Enterobacter,Escherichia, Francisella, Hemophilus, Klebsiella, Morganella, Proteus,Providencia, Pseudomonas, Salmonella, Serratia, Shigella, Vibrio, andYersinia species), spirochete bacteria (e.g., Borrelia species includingBorrelia burgdorferi that causes Lyme disease), anaerobic bacteria(e.g., Actinomyces and Clostridium species), Gram positive and negativecoccal bacteria, Enterococcus species, Streptococcus species,Pneumococcus species, Staphylococcus species, Neisseria species.

In some embodiments, the individual has a neoplastic disease, where aneoplastic disease includes, but is not limited to, leukemia, acuteleukemia, acute lymphocytic leukemia, acute myelocytic leukemia,myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia,chronic leukemia, chronic myelocytic, (granulocytic) leukemia, chroniclymphocytic leukemia, Polycythemia vera, lymphoma, Hodgkin's disease,non-Hodgkin's disease, Multiple myeloma, Waldenstrom'smacroglobulinemia, Heavy chain disease, solid tumors, sarcomas andcarcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor,cervical cancer, uterine cancer, testicular tumor, lung carcinoma, smallcell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, and neuroblastomaretinoblastoma.

In some cases, the individual has, or is at risk of contracting, aparasitic disease. Parasitic diseases that can be treated or preventedby the methods of the present disclosure include, but are not limitedto, amebiasis, malaria, leishmania, coccidia, giardiasis,cryptosporidiosis, toxoplasmosis, trypanosomiasis, schistosomiasis, andfilariasis.

In some cases, the individual has, or is at risk of contracting, afungal disease. Fungal diseases that can be treated or prevented by themethods of the present disclosure include, but are not limited toCandida spp. including C. albicans, Aspergillus spp., Cryptococcus spp.including C. neoformans, Blastomyces sp., Pneumocytes spp., orCoccidioides spp.

In some cases, the individual has, or is at risk of contracting, a worminfection, a fluke infection, etc. Also encompassed are infections byvarious worms, such as but not limited to ascariasis, ancylostomiasis,trichuriasis, strongyloidiasis, toxoccariasis, trichinosis,onchocerciasis filaria, and dirofilariasis. Also encompassed areinfections by various flukes, such as but not limited toschistosomiasis, paragonimiasis, and clonorchiasis.

In some embodiments, the individual has an autoimmune disorder or animmune dysfunction, or is at risk of developing an autoimmune disorderor an immune dysfunction. In some cases, the disease is selected fromthe group consisting of, but not limited to, allergy, rheumatoidarthritis, asthma, diabetes, systemic lupus erythrymetosus (SLE),Grave's disease, atherosclerosis, multiple sclerosis, schizophrenia,Alzheimer's, depression, hypopituitarism, neurodegenerative disorders,cardiovascular diseases, obesity etc.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); i.n., intranasal(ly);i.v., intravenous(ly); s.c., subcutaneous(ly); and the like.

EXAMPLES

Materials and Methods

The following materials and methods were used in the Examples describedbelow.

Materials

AIM V serum-free media was obtained from the Life Technologies(Burlington, Ontario, Canada). Nylon wool was purchased from RobbinsScientific (Sunnyvale, Calif.), made into 10 ml columns, and autoclavedto make it sterile. Ficoll-Paque was obtained from Pharmacia Biotech,(Quebec, Canada). Anti-CD3 (OKT3) antibody was used as purified antibodyobtained from culture supernatant of clones purchased from the AmericanType Culture Collection (ATCC) (Rockville, Md.). PHA was obtained fromSigma Chemical Company. Cytokine kits were purchased from eBioscience(San Diego, Calif.). Poly 1:C, monophosphoryl lipid A (MPL) werepurchased from Sigma Chemical company. Ribavirin was purchased from TRC(Toronto, Canada). Telaprevir was bought from LGM Pharma (TN, USA).Peginterferon was obtained University of Alberta Hospital pharmacy.Wild-type, lipopolysaccharide (LPS)-negative, S-layer negative andrecombinant H5-HA Caulobacter crescentus were grown at room temperature(22-27° C.) in the incubator, and heat-killed at 60-80° C. for 30-60minutes. HEK 293 cells expressing human TLRs or NLRs were purchased fromInvivogen (San Diego, Calif.).

Methods

PBMCs and DCs

Peripheral blood mononuclear cells (PBMCs) were obtained from normalhuman donors using Ficoll-Paque. T cells were purified using nylon woolcolumns. Briefly, 0.75 g of nylon wool was loaded on a 10 ml syringe.The columns were pre-incubated with media for 25 min. and 10⁸ PBMCs wereloaded on the top. After a 45-min. incubation at 37° C., non-adherent Tcells were eluted with warm AIM V media (37° C.). To obtain dendriticcells (DCs), adherent PBMCs were cultured with recombinant GM-CSF andIL-4 for 5-6 days in RPMI media, using procedures well established inthe literature.

Proliferation Assay

PBMCs or enriched T cells were cultured in AIM V media in 96-well flatbottom plate at 2×10⁵ cells per well in the presence or absence ofmitogens (OKT3, 1 μg/ml; or phytohemagglutinin (PHA), 5 μg/ml; orallogeneic irradiated PBMCs 2×10⁵/well). Test compounds were added atvarious concentrations (from 0.00001-100 μg/ml). Stock solutions of testcompounds were made at 10 mg/ml in dimethylsulfoxide (DMSO) andequivalent amount of DMSO was added to stimulated cultures to obtain themedia control. The plates were incubated for 4-5 days at 37° C. inhumidified 5% CO₂, pulsed for final 12-18 hours with 1 μCi/well³H-thymidine in 50 μl AIM V media. The contents of the well wereharvested onto glass fiber filters using a multiple automated sampleharvester, and the ³H-thymidine incorporation was determined by liquidscintillation counter and represented as counts per minute (CPM)incorporated/well. Each group was set up in 3-5 replicate wells and thedata was calculated with mean CPMs of the replicates. Percent controlwas calculated as 100×CPM with the compound/CPM without the compound.

Release of Cytokines

Human PBMCs as purified above were cultured in 24 well plates in AIM Vmedia at 2×10⁶/well/2 ml with or without 0.5 μg phytohemagglutinin(PHA)/well. Test compounds were all added at 10 μg/ml, with anequivalent amount of DMSO added in control wells. The plates wereincubated for 3 days at 37° C. in humidified 5% CO₂ incubator andculture supernatant (1.6 ml) was collected for cytokine analysis byenzyme-linked immunosorbent assay (ELISA). As the negative control,non-stimulated cell supernatant was used. The supernatant was tested forcytokines IL-2, IL-10, IL-17A, IL-22, IL-12, IL-6, IFN-7, IFN-α TNF-α,and granulocyte-macrophage colony stimulating factor (GM-CSF) using R &D Systems (Minneapolis, Minn.) or eBioscience ELISA kits usingmanufacturer's protocol. Each assay was done in duplicate. In theinitial tests, the positive and negative control wells (without testcompounds) were tested in titrating concentrations to determine thedilution of the sups that lies well within the detection range ofstandard cytokine concentrations. This was performed with eachexperiment independently and allowed the experiment to be conducted at adilution range where the standard curve was a straight line and thesecreted cytokines in experimental groups could be accuratelyquantitated. Final cytokine concentration was then determined taking thedilution factor into consideration. Percent of control amount ofcytokines was calculated as 100×pg/ml cytokine in the presence of testcompounds/pg/ml cytokine in the absence of test compounds.

Activation of Human PBMCs and In Vitro Antiviral Activity

Human PBMCs (2×10⁶/ml) from individual normal healthy donors wereincubated with HKCC (10⁵-10⁷/ml) in AIM V media for 24 h at 37° C. in anincubator, followed by collection of supernatants. The supernatant wasthen used at various concentrations (300, 450 or 500 μI for total 1 mlof media) to determine anti-HCV activity as follows. Briefly, 1×10⁵ HCV1a replicon containing Huh-7 cells per well were plated in 24-wellplates in DMEM media supplemented with fetal bovine serum (10%) andselection antibiotic G418. On the next day, replicon cells wereincubated at 37° C. with various volumes of supernatants, indicateddrugs, with or without telaprevir or ribavirin. The cells were incubatedfor five days. After the 5-day incubation period, cells were used forRNA determination. Total cellular RNA was extracted using an RNAeasy-96kit (Qiagen, Valencia, Calif.), cDNA was synthesized using iScript cDNAsynthesis kit (BioRAD, CA) and the copy number of HCV RNA was determinedusing a quantitative polymerase chain reaction (PCR) (Q-PCR) assay usingprobes (BioRAD, CA). β-Actin was used to normalize the HCV RNA copynumbers. The primers used for PCR assays were: HCV UTR F: 5′-CTG TCT TCACGC AGA AAG CG-3′ (SEQ ID NO:1); HCV UTR R: 5′-CAC TCG CAA GCA CCC TATCA-3′ (SEQ ID NO:2); β-actin F: 5′-CGA TGC AGA AGG AGA TCA CTG-3′ (SEQID NO:3); β-actin R: 5′-CGA TCC ACA CGG AGT ACT TG-3′ (SEQ ID NO:4). The% inhibition or HCV RNA copy number/10⁶ cells shown are the averages of3 replicates and the standard deviations were within 10%. Similarly, thesupernatants collected with HKCC treatment of PBMCs can be effectiveagainst the HCV 1b genotype and multiple genotypes (2a, 2b, 3a, 4a, 5aetc.) of HCV.

Inhibition of Mycobacterium

To determine the intracellular inhibition of Mtb and M. avium, humanmonocytic cell line (THP-1) was infected with M. avium or Mtb H37Rausing published procedures, followed by two treatments (on days 0 and 4)with supernatants (50%) collected from human PBMCs treated for 24 hrswith HKCC or PBS in 24 well plates. Supernatants collected from threedifferent donor PBMCs stimulated with HKCC were tested as donors #1, #2and #3. In controls clarithromycin, rifampicin or HKCC were addeddirectly to infected THP-1 cells. Five days after second treatment,THP-1 were collected, lysed and plated on 7H11 agar plates to determinebacterial CFUs.

Adjuvants

Caulobacter crescentus was grown at 25-30° C. in liquid PYE medium (0.2%peptone, 0.1% yeast extract supplemented with 1 ml/L of 20% MgSO₄ and10% CaCl₂) containing 2 μg/ml chloramphenicol. The bacterial cellconcentration was determined as 3.0×10⁹ CFU/ml/1 optical density (O.D.)at 600 nm. The purity of bacterial cultures was examined byphase-contrast microscopy and by plating the bacterial cultures on PYEcontaining solid agar plates. Wild-type Caulobacter crescentus wassuspended in PBS and heat killed at 80° C. for 30-60 minutes in a waterbath, and is referred to as HKCC.

Monophosphoryl lipid A (MPL) was purchased from Sigma (USA). An aqueousformulation containing MPL at a 4:1 molar ratio was prepared as permanufacturer's instructions, aliquoted and stored at 4° C. Polyargininehydrochloride was purchased from Sigma (USA). IFA was purchased fromPierce Biotech (USA).

Antigens/Peptides

Seasonal trivalent influenza vaccine Vaxigrip^(R) (2009-2010 season)containing viral antigens from A/California/7/2009 (H1N1),A/Perth/16/2009 (H3N2) and B/Brisbane/60/2008 was purchased from SanofiPasteur (Canada). Recombinant HBV core was purchased from United StatesBiologicals (MA, USA). Recombinant HCV proteins (NS3, NS3+NS4) were fromChiron Corp. The peptides and lipopeptides were custom synthesized by aGenscript Inc (NJ, USA). The HCV NS3 peptides (1127-46, 1187-1206,1248-71, 1367-86, 1487-1506, 1507-26, 1547-66, 1607-26, 1621-40,1637-57), Influenza M2e peptide (SLLTEVETPIRNEWGCRCNDSSD; SEQ ID NO:5),Mtb Ag85B peptides (68-88, 93-112, 126-142, 143-167, 199-218, 240-251,257-273) and malaria Spf66 peptide were used unmodified or modified atcarboxyl terminal by the addition of a lysine-palmitoyl orlysine-dipalmitoyl group. All peptides/lipopeptides were prepared at 10mg/ml as stock solutions in DMSO and stored frozen. The stocks werediluted with phosphate-buffered saline (PBS) as required. Recombinantadenovector containing HCV-NS3 were prepared according to publishedprocedures.

Mice

6-8 Weeks old C57BL/6 or BALB/c, male or female mice were purchased fromCharles River Breeding Laboratories. All animal experimental protocolsused in this study were approved by the University of Alberta AnimalCare and Use Committee for Health Sciences, and conducted in accordancewith the guidelines of the University of Alberta, Edmonton, Canada.

Viruses, Bacteria, and Tumor Cell Lines

For the surrogate vaccinia-HCV model, the Western Reserve (WR) strain ofvaccinia virus (VV) (1×10⁷ pfu/mouse) containing HCV NS3-NS5 region ofthe HCV BK strain (genotype 1b) [rVV-(NS3-NS5)] was used to challengefemale C57Bl/6 mice intraperitoneally. The virus was grown in BHK-cells.The titer of virus was determined by plaque assay on BHK-cells and wasstored at −80 C.° until use.

In the influenza infection models, the mouse adapted influenza strain ofH1N1 (A/PR8/34) was used intranasally to challenge mice. The viruseswere grown in MDCK cells. The virus was quantified using a cellularELISA protocol detecting the intracellular influenza nucleoprotein (NP).Briefly, MDCK cells were plated at 1×10⁴/well in 96 well tissue cultureplate and allowed to adhere for 24 hours. Biological sample or stock ofthe influenza virus prepared was added on MDCK monolayer and incubatedfor 2 hrs. At this time, plates were washed and cultured for further36-72 hrs, with the last 8 hrs in the presence of Brefeldin A. The cellswere then fixed using formaldehyde and treated with anti-NP antibody andsaponin solution. The intracellular bound anti-NP antibody was detectedby using Goat anti mouse-biotin labeled antibody which was then detectedby Sreptavidin.

In the tuberculosis model, Mycobacterium tuberculosis (Mtb) H37Ra(0.5×10⁶ cfu/mouse) was used intravenously to infect BALB/c female mice.The mice were housed in a specific pathogen-free facility.

For the tumor challenge experiments EL-4 (a C57Bl/6 mouse derived T celllymphoma cell line) and B16 (a C57Bl/6 mouse derived melanoma cell line)(ATCC) were used.

Immunization(s)/Treatments of Mice and Sample Collections

The mice were administered once, twice or thrice with a mixture ofpeptide/lipopeptide/protein antigens with heat-killed (80° C. for 30-60min.) Caulobacter crescentus (HKCC) in PBS using routes as stated indifferent figures.

In prophylactic experiments, HKCC was administered with or withoutantigens to mice intranasally, subcutaneously or orally as described indetail in each figure.

In therapeutic experiments, pre-challenged mice were administered withHKCC with or without antigens intranasally, subcutaneously,intramuscularly, or orally as described in detail in each figure.

The amount of antigens and adjuvant are described in figure legends.Untreated mice were given equivalent volume of PBS or salinecorresponding to the experimental group. After euthanization of mice atspecific times, blood, spleen, inguinal lymph nodes, nasal washes, lungwashes etc. were collected.

Isolation of Splenic T Cells

At specific times after immunization, the mice were euthanized to obtainsplenocytes. The spleens were pooled from 3-5 mice and ground to asingle cell suspension and filtered through a Falcon 100 μm nylon cellstrainer. After centrifugation, the cell pellet was resuspended in 2 mlof sterile distilled water and briefly vortexed. Immediately, 2×PBS wereadded and after a brief vortex the volume was made to 25 ml with 1×PBS.The tube was centrifuged and the cell pellet was resuspended in 10 ml ofcomplete RPMI. It was again filtered through a Falcon 100 μm nylon cellstrainer and centrifuged. The cell pellet was resuspended in 2 ml ofmedia and passed through an equilibrated nylon wool column. The columnwas washed after 45 min of incubation at 37° C. and the flow throughcontained the splenic T cells. These T cells were taken for theexperiment (˜90% CD3+ T cells).

T Cell Proliferation Assay

Proliferative responses of splenic T cells were measured in triplicatecultures in 96-well flat-bottomed microliter plates. A total of 4×10⁵spleen T cells from immunized mice and 4×10⁵ antigen-presenting cells(APCs) (spleen cells from control syngeneic mice irradiated with 18 Gy)were mixed with different concentrations (0.5-10 μg/mL) of eitherrecombinant NS3 protein (c33c, aa 1192-1457 NS3), truncated polyprotein(c200, NS3-NS4, aa 1192-1931) or control superoxide dismutase (SOD) werecultured in RPMI medium (with 10% fetal bovine serum (FBS)) at 37° C.(5% CO₂) for 4 days. In experiments with different exemplary antigens,relative peptide or proteins were used at concentrations described ineach figure.

The cells were pulsed with 0.5 μCi/well [³H]-thymidine (Amersham) for12-18 h and harvested on filter papers (Perkin Elmer). The levels of[³H]-thymidine incorporated into the DNA of proliferating cells werecounted in a Microbeta Trilux liquid scintillation counter (PerkinElmer). Proliferation is represented as the mean cpm±SE (standard error)of triplicate cultures.

ELISpot Assay for GrB Producing CD8+ T Cells

Enzyme-linked immunosorbent spot (ELISPOT) assay kits were obtained fromR&D Systems and manufacturer's instructions were followed for theELISpot assay. Briefly, 96-well nitrocellulose plates were coated with acapture anti-mouse Granzyme B (GrB) antibody overnight at 2-8° C.,followed by washing the plates for 2-3 times with PBS. After blockingwith the blocking buffer at room temp for 2 hrs, the buffer wasaspirated and mouse splenocytes activated for 3 days as described in Tcell proliferation assay in the presence or absence of antigens wereadded at 1-5×10⁵/well in RPMI media. The plates were incubated at 37° C.for overnight, followed by washing with PBS for 4 times. Plates werethen added with detection antibody and incubated overnight at 2-8° C.,followed by the addition of Streptavidin-alkaline phosphatase (AP) andincubation for 2 hrs at room temperature. Color was developed by addingchromogen 5-bromo-4-chloro-1H-idol-3-yl/nitro blue tetrazolium(BCIP/NBT) for 5-15 minutes. Plates were rinsed with distilled water anddried before enumerating the number of spots/well using an ELISPOTreader Biosys.

Evaluation of Antibody Responses

The levels of antibodies (IgG, IgG1, IgG2a, IgA) in serum, lung andnasal washes were determined using enzyme-linked immunosorbent assays(ELISAs). Briefly, 96-well nitrocellulose (Nunc) plates were coated withrelevant antigen (such as OVA, influenza, Hepatitis B surface antigen(HBsAg), HBV core antigen, whole cell lysate from EL-4 or B16) andincubated overnight at 4° C. The plates were blocked with PBS containingnormal mouse serum, followed by incubating with the experimental samplesat different dilutions for 2 hrs at room temperature. After washing theplates for 4 times, Anti mouse Ig isotype antibodies conjugated withAlkaline phosphatase (AP) were added, followed by incubation for 2 hrs.After washing the plates, PNPP substrate was added and color developmentwas read on Fluostar ELISA reader at 405 nm wavelength. All reagents forantibody detection were obtained from Southern Biotech (Birmingham,Ala.).

Flow Cytometry Analysis of Surface Markers, Intracellular Granzyme B andFoxp3.

A total of 5×10⁵ cells from immunized mice were taken for intracellularand extracellular staining with multicolor fluorescently-labeled mAbs(concentrations according to manufacturer's instructions). The cellswere incubated with Fc mouse-serum (Sigma) to prevent non-specificbinding and washed with fluorescence-activated cell sorter (FACS)-buffer(2% fetal bovine serum in 1× phosphate-buffered saline (PBS). Afterincubation for 30 minutes with anti-mouse CD3e-FITC, CD4-PECy-5,CD25-PE-Cy7, CD8a-APC-Cy7, anti-PD-1-PerCP eFluore 710,anti-CD49b-Alexafluor-700 (for BALB/c and C57bl/6 mice) or anti-NK1.1(for C57bl/6 mice), anti-CD11c, anti-CD19, anti-CD11b, anti-CD40,anti-CD69, anti-CD25 etc. (eBioscience) for extracellular markers at 4°C., the cells were washed twice and fixed in fixative solution (1%paraformaldehyde in FACS-buffer) for 5 minutes. After washing twice, thecells were incubated with cold permeabilization buffer (FACS-buffer+0.3%Saponin (Sigma)+5% normal human serum in PBS) for 5 minutes followed byaddition of anti-GrB-PE (Caltag Laboratories, Burlingame, Calif.) andanti-Foxp3-APC (eBioscience) and further incubated for 30 minutes at 4°C. The cells were washed once with FACS-buffer containing 1 Saponin andfixed. They were read in FACS-Canto and analyzed using FACS-DIVAsoftware (Becton Dickinson, Mountain View, Calif.). Each marker wasgated based on its respective isotype-matched control monoclonalantibodies. Similar staining methodology was used with human DCs todetermine various activation markers (CD11c, CD80, CD86, DEC-205 etc.)(eBioscience) in flow cytometry experiments.

Mouse Cytokine ELISA

Cytokines secreted in the supernatant of proliferating co-cultures, ormouse serum samples were measured using sandwich ELISA kits followingthe manufacturer's protocol (eBioscience, CA, USA) for the presence ofIL-10, IL-12, GM-CSF, IL-17A, IFN-γ, IFN-α, IFN-β, IL-2, TGF-β and IL-4.A dilution of 1:2 to 1:50 was used for the samples with the standardsranging from 5 to 2000 pg/ml. Finally the ELISA plates were read and theconcentrations were calculated with an automated ELISA plate reader(Fluostar Optima, BMG Labtech).

Quantitation of Vaccinia Virus Expressing HCV Genes in Ovaries by PlaqueAssay

Mice were sacrificed 5 days post infection. Pairs of ovaries fromindividual mouse were harvested and homogenized in 1 ml DMEM. They weretreated with 3 repeats of thaw freeze cycle, sonicated and centrifugedat 3500 rpm for 15 min, the supernatant were stored at −80 C.°, untilused for plaque assay or virus nucleic acid isolation. To determinevirus titer, 10-fold dilutions of supernatant were plated onto BHK-21cells in 6 well plates. After 90 min of incubation at 37 C.° in 5% CO₂and then 2.5% DMEM was applied. The plates were incubated for 48 hoursat 37 C.° in 5% CO₂. The cells were then stained with 0.1% crystalviolet in 20% ethanol and the number of plaques was enumerated.

CFU Assay to Determine Mtb Load in Various Organs

Three weeks after H37Ra infection, mice were euthanized and lung, liverand spleen were removed aseptically and individually homogenized. CFUcounts per 10% organ were determined on 7H11 selective agar platespurchased from BD Biosciences. The plates were incubated at 37° C. inambient air for up to 3-4 weeks prior to counting the colonies.

Results

The following examples are intended to illustrate rather than limit thescope of the invention.

The adjuvant and immunotherapeutic effects of HKCC were tested withvarious types of vaccines in different models and indications viasystemic and mucosal routes as follows.

Example 1: Heat-Killed Caulobacier crescenius (HKCC) as a MucosalAdjuvant

The effect of HKCC on an immune response against a whole protein antigenwas investigated using ovalbumin (OVA) upon intranasal immunization.C57/b16 male mice were immunized once intranasally with a mixture of OVAantigen (50 μg/mouse) and HKCC at three different doses (0.5-50×10⁶colony forming units (CFU)/mouse, indicated as 0.5M, 5 M and 50 M,respectively). The mice were euthanized 8 days after single immunizationand OVA specific T cell proliferation response was examined using 3H-Tdrincorporation assay (FIG. 1).

The results obtained indicate that HKCC induces T cell responses againstOVA upon single intranasal (mucosal) administration. Dose response studysuggests that HKCC at 50×10⁶ CFU/mouse induces higher antigen specific Tcell responses following single intranasal immunization as compared tothe lower doses (0.5-5×10⁶ CFU/mouse) (FIG. 1).

Example 2: Effect of Live Caulobacter crescentus (CC) and HKCC onAntigen-Specific Immune Responses Against Ovalbumin (OVA)

The effect of live and heat-killed CC for the induction of OVA-specificcellular and humoral immune responses in vivo was determined. Groups offive C57/b16 mice were immunized by the subcutaneous (s.c.) route at thebase of the tail twice on days 0 and 14, or once with live CC or HKCC at50×10⁶ CFU/mouse and OVA antigen (20 μg/mouse) in 100 μl totalvolume/mouse. Mice were euthanized 8 days after one immunization or 2weeks (wks) after two immunizations. T cell proliferative responsesagainst OVA antigen were determined from T cells obtained from spleenand lymph nodes (FIG. 2A, 2B, 2D). Antigen specific GrB producing CTLswere quantified (ELISPOT assay) using splenocytes (FIG. 2C). Serumantibody (IgG and IgG1) responses was measured using ELISA (FIG. 2E).

Example 3: HKCC as an Adjuvant for Therapeutic HBV Vaccine to InduceCellular and Humoral Immune Responses

C57/b16 male mice were immunized twice (at 14 days intervals) with amixture of recombinant HBV core antigen (5 μg/mouse) and HKCC (50×10⁶CFU/mouse) by intranasal route. Mice were euthanized 1 week and 3 weeksafter second immunization. Spleen, blood, lung washes were collected andused to determine cellular and humoral immune responses against HBV coreantigen. Splenocytes obtained from mice immunized with HBV core and HKCCshowed much higher T cell proliferation, IFN-gamma and IL-12 productionand Granzyme B (GrB)-producing CTLs compared to HBV core aloneimmunization (FIG. 3A-F). In addition, systemic IgG and IgG2a, as wellas mucosal (lung) IgG and IgA against HBV core antigen were induced togreater extent in mice immunized with HBV core plus HKCC compared to HBVcore antigen only both in the short and the long-term (1 vs. 3 week post2nd immunization) (FIG. 3G-L).

These results demonstrate that HKCC induced strong and long-lastingantigen specific T cell (CD4+, CD8+) and antibody responses against theconserved HBV core antigen in an animal model as shown in FIG. 3A-L.These studies suggest that HKCC can adequately induce both cellular andhumoral immune responses against HBV antigen and could be used astherapeutic vaccine regimen. Also, these results suggest that aneedle-free nasal immunization with an antigen using HKCC as an adjuvantcould be an effective approach of vaccination.

Example 4: HKCC Exhibits Potent Adjuvant Activity Enhancing HCV DerivedNS3 Specific T Cell Responses

In the case of HCV infection, failure to generate and maintain aneffective cellular immune response in the acute phase is likelyresponsible for the high rate of chronicity. A vaccine that can induceefficient T cell responses against conserved antigens of HCV or animmunotherapeutic approach that can induce broad and effective anti-HCVimmunity would be beneficial for the prevention and/or treatment of HCVinfections.

In order to determine if HKCC could be used as an adjuvant for HCVvaccine, C57Bl/6 female mice were immunized subcutaneously twice (at 10day intervals) with a mixture of 10 different lipopeptides (NS3 1248-71,1621-40, 1127-46, 1187-1206, 1367-86, 1487-1506, 1507-26, 1547-66,1607-26, 1637-57, 2.5 μg each peptide/mouse) and HKCC (50×10⁶CFU/mouse). The mice were euthanized 15 days after second immunization.The spleens of immunized mice were isolated and examined for antigenspecific proliferative responses against recombinant NS3 antigen orcontrol antigen SOD (FIG. 4). The results demonstrated that HKCCadjuvant greatly enhances HCV antigen specific T cells when compared tounimmunized mice. Mice immunized with NS3 peptides only, did not showNS3 specific T cell responses and were comparable to unimmunized groups.These results also suggest the applicability of HKCC adjuvant forpeptide-specific subunit vaccines.

Example 5: HKCC as an Adjuvant for Subunit Vaccine for Tuberculosis:Reduction of Mycobacterial Load in Lungs, Liver and Spleen

In order to determine if HKCC could be used as an adjuvant to inducemycobacterial antigen specific immune responses which could lead toanti-mycobacterial immunity and reduction in bacterial load, groups offive mice were immunized twice subcutaneously at 12-day intervals with amixture of 7 monolipopeptides derived from Ag 85B (68-88, 93-112,126-142, 143-167, 199-218, 240-251, 257-273, 5 μg each peptide/mouse)and HKCC (50×10⁶ CFU/mouse). The immunized mice were challengedintravenously with 0.5×10⁶ cfu/mouse Mth H37Ra six weeks after secondimmunization. Infected mice were euthanized three weeks after Mtbchallenge. Lungs, liver and spleen were collected from individual miceand used for CFU assay to determine bacterial load in these organs (FIG.5A-C). The CFU data obtained from five individual mice demonstrated thatpreimmunization with Mtb Ag85B derived peptides along with HKCCpartially protects mice from getting Mtb infection and/or leads to muchlower bacterial loads in lungs, liver and spleen, compared tounimmunized mice (FIG. 5A-C). Mice immunized with Ag85B peptides only,did not show a reduction in the bacterial loads compared to unimmunizedgroups (data not shown). These results demonstrated that HKCC as anadjuvant to a subunit vaccine can provide efficient protection againstbacterial infection and/or reduces bacterial load upon infection (FIG.5A-C). Further, HKCC represents a novel potent immunostimulator inproviding systemic protection against lung, liver and spleen infectionfrom Mtb upon immunization with peptide-based antigens of amycobacterial protein. Similarly, HKCC can provide strong protectionwhen combined with other immunostimulatory proteins produced and/orsecreted by the TB bacterium.

Example 6: HKCC as an Adjuvant for Tumor Vaccine: Reduction of EL-4Tumors after Single s.c. Immunization

Whole tumor cell vaccines are interesting candidates for cancer vaccinesas they could provide protection mediated by multiple antigens' specificT cells and B cells as opposed to single antigen or subunit basedvaccines. However, these strategies are limited due to insufficientimmunity induced in response to whole tumor cells. In order to examineif HKCC can bolster immune responses to whole tumor cells, groups offive C57Bl6 mice were immunized once subcutaneously with a mixture ofirradiated EL-4 cells (1×10⁶/mouse) and HKCC (50×10⁶ CFU/mouse). Theimmunized mice were challenged with 0.25×10⁶ EL-4 cells/mouse in 100 μlPBS subcutaneously in the lower left flank eight days after theimmunization. Tumor growth was measured for 28 days after challengeusing digital calipers in two perpendicular directions, and mice werehumanely euthanized. Tumor area were calculated as length×width (in mm).The results showed that administration of irradiated tumor cells withadjuvant HKCC generated significant protective effect against a solidtumor in vaccinated mice with robust systemic tumor specific T cell andantibody responses (FIG. 6A-D). In addition, HKCC as an adjuvant in awhole irradiated tumor cell vaccine inhibits tumor progression (FIG. 6A,B).

Example 7: HKCC as an Adjuvant for Lung Cancer Vaccine: Reduction inLung Metastases after Single s.c. Immunization

To examine if HKCC can bolster immune responses to whole tumor cells ina metastatic lung cancer model, groups of five C57Bl6 mice wereimmunized once subcutaneously with a mixture of irradiated B16 cells(1×10⁶/mouse) and HKCC (50×10⁶ CFU/mouse). The immunized mice werechallenged with 0.4×10⁶ B16 cells/mouse in 50 μl PBS intravenously inthe tail vein eight days after immunization. Mice were humanelyeuthanized 12 days after tumor challenge. The lungs and serum werecollected. Lung nodules were examined by pictures taken and weight oflungs were determined (FIG. 7A, B). Further, sera were tested for thepresence of IgG specific against B16 cell lysate (FIG. 7C). The resultsobtained demonstrated that HKCC adjuvanted whole irradiated tumor cellvaccine (whole irradiated tumor cell in a formulation with HKCC as anadjuvant) inhibits metastatic lung cancer progression and induce tumorspecific antibody responses (FIG. 7A-C).

Example 8: Antitumor Activity of HKCC Against B16 Melanoma LungMetastasis after Subcutaneous Treatments

In a cancer or tumor bearing individuals, usually the presence ofspecific tumor antigens lead to priming of antigen specific immuneresponses, however, they are not effective in providing protection fromtumor progression due to several mechanisms regulating and/or inhibitingthese immune responses. To determine if treatment with HKCC as animmunomodulator could provide reduction in cancer progression, groups offour C57Bl6 mice were challenged with 0.4×10⁶ B16 cells/mouse in 100 μlPBS intravenously in the tail vein. Starting from day 3 post challengewith B16 melanoma cancer cells, HKCC (50×10⁶ cfu/mouse) was administeredtwice subcutaneously at one week interval. Three days after the lasttreatment, mice were euthanized. Lungs and serum were collected. Theresults obtained demonstrate that immunotherapy with HKCC alone leads tomarked reduction in metastatic lung cancer progression (FIG. 8A),normalization of lung weights (FIG. 8B), and induction of tumor specificsystemic IgG responses (FIG. 8C). No metastasis of B16 cells was seen inother organs. In addition, B cells, DCs, NK and NKT cells were activatedand/or increased significantly in spleens and lungs in treated micecompared to untreated B16 tumor bearing mice (data not shown). Furtherto increasing effective immunity in tumor bearing mice, HKCC therapyalso modulated the percentage of Tregs in both lungs and spleens.Therefore, HKCC therapy results in marked inhibition of lung cancer in ahighly aggressive metastatic mouse model of cancer.

Although this example was based on cancer immunotherapy with HKCC alone,there are other immunotherapeutics and anticancer agents which could becombined with HKCC to further increase the antitumor effect. The presentdisclosure represents HKCC as an attractive therapeutic treatment for arange of cancers at a particular site or a metastasis.

Example 9: Antitumor Activity of HKCC Against a Solid Tumor

To examine whether HKCC can bolster immunity in such a way that itprevents and/or inhibits tumors from growing in the body, animmunotherapy model was used. Groups of five C57Bl6 mice were challengedwith 0.25×10⁶ EL-4 cells/mouse in 100 μl PBS s.c. in the lower leftflank. Six days after, mice were treated once weekly subcutaneously forthree times with HKCC (50×10⁶ CFU/mouse) or PBS control. Tumor growthwas measured for 28 days after challenge using digital calipers in twoperpendicular directions, and mice were humanely euthanized. Tumor areawere calculated as length×width (in mm). Strikingly, treatment with HKCCresulted in significant reduction in tumor progression (FIG. 9A). Inaddition to decreasing tumor burden, HKCC therapy led to decreased PD-1expression on CD4+, CD8+ and NKT cells (FIG. 9B), suggesting thatimmunotherapy with HKCC alone may function through modulating immunemechanisms. These studies suggest that HKCC can be used as immunotherapyto prevent, treat or ameliorate metastasis or recurrence or inhibit thegrowth or proliferation of a variety of cancer cells or tumors in thespecific organ or tissue of an individual improving patient survivalrates. Further, HKCC treatment may also be undertaken to rid the body ofresidual tumor after chemo, radiation or surgical treatments etc.

Example 10: Effect of Live CC and HKCC: HKCC Induces RobustAntigen-Specific Cellular (CD4+ and CD8+ T Cells) Immune ResponsesAgainst TIV (Seasonal) Influenza Vaccine Upon Single Mucosal (i.n.)Immunization with a Low Dose of Antigen

The current seasonal influenza vaccine is assumed to work throughinducing antibody responses against specific variants of influenzaantigens HA and NA. However, literature suggests heterologous protectionin certain instances possibly through induction of cellular immuneresponses and humoral responses against conserved regions of HA presentin the current trivalent vaccine (TIV). Therefore, we examined whetherHKCC can allow strong cellular immunity to be induced in mice immunizedwith HKCC and influenza TIV vaccine. Further, we sought to examine theeffects of live vs. heat-killed CC to function as an efficient adjuvantinducing cellular immunity against influenza vaccine. Groups of fiveC57/b16 mice were immunized by the intranasal route with live CC or HKCCat 50×10⁶ CFU/mouse with Vaxigrip (1.6 μg/mouse) in 30 μl totalvolume/mouse. In the control no adjuvant group, Vaxigrip (1.8 μg/mouse)alone was administered subcutaneously. Mice were euthanized 8 days afterimmunization. Intriguingly, the results obtained demonstrated that HKCCstimulates robust cell mediated CD4+ and CD8+ T cell immunity(proliferation and GrB production) against seasonal flu vaccine(Vaxigrip) as compared to live CC or no adjuvant group (FIG. 10A,B).Therefore, the HKCC adjuvanted vaxigrip (vaxigrip in a formulation withHKCC as an adjuvant) was superior to non-adjuvanted vaxigrip or live CCadjuvanted vaxigrip in inducing cellular immune responses. In contrast,T cell response to a mitogen ConΛ was similar in all three groups (FIG.10A).

These results indicate that intranasal co-administration of HKCC with alicensed trivalent vaccine (vaxigrip) containing a mixture ofhemagglutinin and neuraminidase antigens from H1, H3 and B strains ofinfluenza virus significantly increase antigen-specific CD4+ T cells aswell as GrB producing cytotoxic T cell responses in mice within 8 daysof single immunization. Interestingly, only mice vaccinated intranasallywith HKCC produced dramatic cellular immune responses compared withthose of mice immunized intranasally with live CC or vaxigrip alone.CD8+ T cells are critical in controlling and eliminating respiratoryinfections, especially those caused by highly pathogenic strains ofinfluenza viruses. CD8+ T cells specific for conserved or cross-reactiveepitopes have been shown to mediate heterosubtype cell-mediated immunityagainst influenza strains that differ in HA serotypes. Importantly, Tcell responses generated upon intranasal immunization of mice with HAand NA containing proteins admixed with HKCC, unlike vaxigrip, conferredsignificant protection against intranasal challenge with a heterologousstrain of influenza virus as described in FIG. 14. Thus, HKCC inducesbroadly cross-reactive T cell immunity and would improve the efficacy ofa commercially available influenza vaccine.

Example 11: HKCC Induces Long-Lasting Humoral and CellularAntigen-Specific Cellular Immune Responses Against Co-Administration ofMultiple Antigens of Influenza Upon Mucosal (i.n.) Immunizations withLow Doses of Antigens

The shortcomings with current influenza vaccine are due to the inductionof virus variant specific antibody responses, leading to a necessity ofchanging and updating the vaccine preparation for every influenzastrain. A universal vaccine targeting multiple and conserved antigens ofinfluenza and inducing both cellular and humoral immunity against theseantigens will be an important step forward towards the development of anuniversal influenza vaccine. Therefore, we examined if HKCC could beused as an efficient adjuvant with multiple influenza antigens (M2e, andHA and NA containing Vaxigrip) to induce both humoral and cellularimmune responses against these antigens.

Groups of five C57/b16 male mice were immunized with a mixture ofseasonal TIV influenza vaccine (Vaxigrip 1.8 μg/mouse), M2e-monolipopeptide (20 μg/mouse) and HKCC (50×10⁶ CFU/mouse) once or twiceintranasally (at 21 days interval). Also, HKCC was used in combinationwith other adjuvants e.g., MPL (a TLR-4 agonist) (5 μg/mouse) or apolymeric compound e.g., poly-L-arginine hydrochloride (100 μg/mouse).Sera samples were collected 8 and 28 days after both single and twoimmunizations and mice were euthanized 8 days or 28 days after twoimmunizations.

The results showed that HKCC induces early, robust and long-lastingantigen specific T cell and antibody responses following one or twointranasal immunization(s) against both TIV and M2e antigens (FIG.11A-G). Importantly, HKCC as an adjuvant in nasal vaccination inducedantigen specific IgA in nasal and lung lavages of the mice (FIG. 11B-E).No IgE were developed against any antigens at both early and later timepoints. Intriguingly, the adjuvant effect of HKCC was furtherpotentiated by the addition of MPL as well as poly-L arginine (FIG.11A-G).

These results show that HKCC can induce robust humoral and cellularimmune responses against a weakly immunogenic antigen, and combiningwith other adjuvants or molecules can further improve the immunogenicityof weak antigens. M2e, the ectodomain of the M2 protein found on thesurface of influenza A viruses, is the most highly conserved surfaceprotein of the virus. The admixing of HKCC induces robust immuneresponses against M2e as well as HA and NA (present in vaxigrip), andprovides potential for a universal vaccine candidate for influenza.Also, HKCC and its combination with other adjuvants/molecules can allowthe generation of strong cellular and humoral immunity simultaneouslyagainst multiple antigens even in a single vaccination. These studiesfurther demonstrate that HKCC can provide enhanced antigen-specificimmune responses when combined with other immunopotentiators to increasethe immunogenicity and reduction of dose of antigens.

Example 12: HKCC Mediated PRR Signaling

Evaluation of the ability of HKCC to stimulate various innate pathogenrecognition receptors (PRRs) (TLR2/6, 3, 4, 5, 7, 8, NOD-1 and NOD-2)was performed using HEK293 cells stably expressing individual TLR orNLR. The cell lines were incubated for 24 hrs at 37° C. with HKCC atdifferent concentrations (10⁴-10⁸ cfu/ml) and TLR/NLR specific ligands(LPS, heat killed E. coli, resiquimod, FSL-1, poly I:C, M-triDΛP) ascontrols. NF-κB activation was determined by measuring the SEAP secretedin to the cell culture media using Quanti blue reporter assay (usingreagents and protocols provided by Invivogen). Surprisingly, the resultsindicate that HKCC does not activate TLR-4 and TLR-5 signaling, butactivates TLR-2/6, 3, 7, 8, NOD-1 and NOD-2 (FIG. 12).

In addition to innate and adaptive immune cells, mammalian pluripotentstem cells (CD34⁺ progenitors) also express various TLRs such as TLR2/6, 7, 8, 9. Therefore, HKCC can stimulate TLRs on stem cells to inducetheir proliferation, differentiation and restoration of homeostasis.

Example 13: S-Layer Negative HKCC Induces Antigen-Specific T CellResponse Against Multiple Antigens of Influenza Upon Mucosal (i.n.)Immunization

To determine the role of surface S protein of CC in providing adjuvanteffects, we used S-layer negative HKCC as an adjuvant in thisexperiment. C57/b16 male mice were given two intranasal immunizations at21 day interval with a mixture of seasonal TIV influenza vaccine(Vaxigrip 1.8 μg/mouse), M2e-monolipo peptide (20 μg/mouse) and S-layernegative HKCC (50×10⁶ CFU/mouse). Mice were euthanized 8 days after thesecond immunization and T cell proliferative responses were examined invitro against Vaxigrip and M2e antigens (FIG. 13). Interestingly theS-layer negative HKCC also provided a bolstering effect on T cellresponse against both of the influenza antigens, similar to thoseobtained using wild-type HKCC, suggesting that the S layer of CC is notessential in its activity as an adjuvant.

Example 14: Enhancement of Spectrum of Protection of Seasonal Flu TIVVaccine (Vaxigrip) Upon Single Mucosal or s.c. Immunization: Protectionfrom Heterologous Virus Infection

Groups of three BALB/c female mice were immunized by the intranasal (1.8μg/mouse Vaxigrip) or subcutaneous (3.6 μg/mouse Vaxigrip) routes withHKCC (50×10⁶ CFU/mouse) in 30 and 100 μl total volume/mouse,respectively. In the control no adjuvant group, Vaxigrip (3.6 μg/mouse)alone was administered subcutaneously. Eight days after immunization,mice were challenged intranasally with 30 μl/mouse of stock of H1N1(PR8) virus and daily weights of individual mouse were recorded. Fourdays after infection, mice were euthanized and viral titers weredetermined in lung homogenates. Bronchoalveolar lavage (BAL) was alsocollected to determine cytokine and infiltrating immune cells.Interestingly, HKCC as an adjuvant in a seasonal flu vaccine (vaxigrip)provided protection from heterologous viral (H1N1) infection as well asprotection from weight loss and enhanced infiltration of innate (DCs,NK, NKT) and adaptive immune cells (CD4 and CD8 T cells) and IL-2production in the lungs upon single i.n. or s.c. immunization ascompared to Vaxigrip alone or unimmunized groups (FIG. 14A-D). Theseresults suggest that HKCC as an adjuvant in a influenza vaccine canprovide cross-protection against unmatched viruses. These resultsdemonstrate effect of HKCC combined with HA and NA proteins as a mucosaland parenteral adjuvant for induction of adaptive and innate immuneresponses, and cytokine, and protection against heterologous influenzavirus. Thus, HKCC has potential to increase the breadth of protectivehetero-subtypic immunity of HA and NA containing existing influenzavaccines. These results also show that single i.n. or s.c. vaccinationwith commonly available influenza vaccine in combination with HKCCstrongly improves the efficacy of a commercially available influenzavaccine.

Although this example was based on influenza TIV vaccine, there areother influenza vaccines such as live attenuated influenza virus vaccineand tetravalent vaccine, which could be used in a similar manner.Therefore, the present disclosure represents attractive target for arange of influenza vaccines.

Example 15: Single Subcutaneous Immunization of a Poorly ImmunogenicAntigen (M2e) Adjuvanted with WT-HKCC or LPS-Negative HKCC Protect fromWeight-Loss after Influenza Virus Infection

To determine the role of lipopolysaccharide (LPS) of CC in providingadjuvant effects, we used LPS negative HKCC as an adjuvant in thisexperiment. Groups of five BALB/c female mice were immunized oncesubcutaneously with M2e peptide or lipopeptide (25 μg/mouse) and WT HKCCor LPS-negative HKCC (50×10⁶ CFU/mouse) in 100 μl total volume/mouse atthe base of the tail. Eight days after immunization, mice werechallenged intranasally with 30 μl/mouse of stock of H1N1 (PR8) virusand daily weights of individual mouse were recorded. Four days afterinfection, mice were euthanized and BAL was collected to determineinfiltrating cells. Immunization with both WT-HKCC or LPS-negative HKCCadjuvanted M2e (M2e in a formulation with HKCC as an adjuvant) partiallyprotected mice from weight loss due to influenza infection and enhancedinfiltration of innate and adaptive immune cells (CD11c⁺ DCs,CD11c⁺CD40⁺ DCs, NKT, CD3⁺CD4⁺ T cells, and CD3⁺CD8⁺ T cells) in thelungs as compared to unimmunized group (FIG. 15A,B). These resultsdemonstrate that HKCC as well as LPS⁻ HKCC are potent inducer of innateand adaptive immune responses and provide protection from influenzavirus infection. This experiment clearly suggested that LPS molecule ofCC is not an essential component for the adjuvant activity of HKCC.

M2e is the most highly conserved surface protein of influenza viruses,therefore, HKCC adjuvanted M2e vaccine is expected to be effectiveagainst highly pathogenic strains of influenza such as H5N1, H7N9 etc.Several findings suggest the protective role of cellular (CD4+ and CD8+T cells) and innate immune responses in other RNA viruses such as Denguevirus (DENV). Therefore, HKCC adjuvanted vaccines by inducing cellularimmune responses may offer protection against other RNA viruses such asDengue virus, West Nile virus, Japanese encephalitis virus, Yellow fevervirus etc.

Example 16: Prophylactic Immunotherapy: Viral (H1N1) Protection UponPre-Treatment (24 hr Before Infection) with HKCC by Parenteral orMucosal Route

To determine if HKCC by virtue of its immunomodulatory activity canactivate immune responses such that an individual is protected fromgetting an infection, we performed prophylactic treatment experimentwith HKCC only given by various routes. Groups of five BALB/c femalemice were treated with HKCC (50×10⁶ cfu/mouse) by subcutaneous (100 μlvolume/mouse at the base of the tail), intranasal (30 μl total/mouse) ororal route (100 μl volume/mouse). Twenty four hours after treatment,mice were challenged intranasally with 30 μl/mouse of stock of H1N1(PR8) virus and daily weights of individual mouse were recorded. Two andfive days after infection, mice were euthanized and BAL and lung sampleswere collected. In this experiment, prophylactic treatment with HKCCprotected mice from weight loss due to influenza infection, reducedviral load, enhanced infiltration of innate and adaptive immune cellsand induction of IL-2, IFN-g, IL-17A cytokines in the lungs (FIG.16A-D).

Significant amount of IL-2 and appreciable levels of IFN-γ and IL-17Awere detected in the lungs of mice treated with HKCC as compared tountreated mice after influenza infection. Also, cytotoxic T lymphocytes,NK, NKT and DCs were detected in BAL in the HKCC treated mice withoutany inflammation in the lungs. These data suggest that HKCC is a potentinducer of TH1, TH17, NK, NKT and CD8 T cell responses and useful in thetreatment of viral infections. Altogether, this study demonstrates thatHKCC is a safe and effective immunotherapeutic agent that can provideantiviral therapeutic effect by all s.c., i.n. and oral routes againstinfluenza and other respiratory viruses such as RSV, SARS etc.Generation of adaptive and innate immune responses to fight anestablished influenza infection is very important to combat influenza inthe elderly. IL-17A producing T cells have been shown to be protectiveby others in influenza infection, implying the importance of TH17response in immunity against pathogens.

Example 17: Antiviral Activity of HKCC Against H1N1 Infection:Immunotherapeutic Effect

To determine if HKCC by virtue of its immunomodulatory activity, can beused as an immunotherapeutic agent to treat and/or ameliorate aninfection, we performed immunotherapy experiment where mice were treatedwith HKCC alone 24 hrs after infection with influenza virus. Groups offive BALB/c female mice were challenged intranasally with 30 μl/mouse ofstock of H1N1 (PR8). Twenty-four hours after the infection, mice weretreated with HKCC (50×10⁶ cfu/mouse) subcutaneously (100 μl volume/mouseat the base of the tail), intranasally (30 μl total/mouse) or orally(100 μl volume/mouse, once or thrice). Five days after infection, micewere euthanized and lungs were collected to determine viral titers. Theadministration of HKCC in mice infected with H1N1 reduced viral (H1N1)load using subcutaneous, intranasal and oral routes (FIG. 17). Thesestudies suggest that HKCC can be used effectively in a therapeuticregimen for the treatment of diseases by various routes. These resultsalso demonstrate that HKCC could be used to treat other RNA and/orrespiratory viruses.

Example 18: Antiviral Activity: HKCC Induces Cytokines from Human PBMCswhich can Inhibit HCV Replication Alone and in Combination with OtherAntiviral Drugs in Huh-7 Replicon Containing Cells

Single treatment of Huh-7-1a replicon containing cells with supernatantsfrom HKCC treated PBMCs from different individual donors for five daysresulted in sustained reduction of viral RNA without affecting thecellular RNA (FIG. 18A,B). There was no significant difference in HCVlevels with untreated PBMCs' supernatant. The supernatant was alsotested to assess the potential use of HKCC in combination therapies withother anti-HCV chemotherapeutic agents telaprevir and ribavirin. Theresults obtained demonstrate that combination treatment of repliconcells with HKCC and inhibitors targeting HCV protease or other pathwayslead to synergistic antiviral effects (FIG. 18B). As control fornon-specific effects, HKCC was also tested directly to HCV repliconcells where it did not have any effect on HCV replication. These datasuggest that HKCC's activity is due to the induction of antiviralcytokines and thus it can be used to treat HCV infection of differentgenotypes, IFN-α non-responder HCV, drug-resistant HCV and other hard totreat HCV populations including patients with co-infections and cancers.

Although this example was based on activity against HCV, there are otherviruses and microorganisms that can be inhibited by soluble factorsproduced by PBMCs stimulated with HKCC alone and/or in combination withother chemo and immunotherapeutics. Therefore, the methods of thepresent disclosure represent an attractive treatment for a range ofinfections.

Example 19: Intracellular Activity: HKCC Induces Cytokines from HumanPBMCs which can Inhibit Intracellular Bacterial Replication

Human monocytic cell line (THP-1) was infected with M. avium or MtbH37Ra, followed by two treatments (on days 0 and 4) with supernatants(50%) collected from human PBMCs treated for 24 hrs with HKCC or PBS.HKCC efficiently inhibits intracellular mycobacterial growth in ahost-immune dependent manner as compared to supernatants collected fromPBS treated human PBMCs (FIG. 19). These data provide supportiveevidence that HKCC has strong potential to efficiently inhibit growth ofother intracellular pathogens such as mycobacteria, lysteria,leishmania, intracellular G⁺ and G⁻ bacteria and malaria parasites etc.

Example 20: HKCC is Effective in Combination with Chemotherapeutic Drugin Reducing Bacterial Burden

Groups of 5 BALB/c female mice were challenged with H37Ra (0.5×10⁶cfu/mouse) intravenously. Five days post infection, mice were treatedwith HKCC (s.c.), and INH (oral) or PBS using a schedule shown in theFIG. 20. HKCC in combination with first-line tuberculosis drug INHprovides enhanced antimycobacterial effects in a Mtb infected mousemodel than INH alone. Significantly higher reduction in mycobacterialloads was observed in lungs, liver and spleen as compared to INH and notreatment groups (FIG. 20). Thus, HKCC can be combined with anantimicrobial agent to achieve more complete inhibition of a pathogen,shorten the treatment duration, reduce the dose of chemotherapeuticagents and also treat drug resistant strains of pathogens, as animmunotherapeutic.

Example 21: HKCC Elicits Potent Adjuvant Activity Enhancing MalariaDerived Antigen Spf66-Specific T Cell Responses

To determine if HKCC could be used as an adjuvant for Malaria vaccine,male C57/b16 mice were immunized subcutaneously twice (at 12 daysinterval) with HKCC (50×10⁶/mouse)+Spf66 peptide (20 ug/mouse), Spf66peptide (20 ug/mouse) alone or PBS. Mice were euthanized eight daysafter second immunization. The spleens of immunized mice were isolatedand examined for antigen specific proliferative responses. The resultsdemonstrated that HKCC adjuvant enhances malaria antigen-specific Tcells when compared to peptide alone or unimmunized mice (FIG. 21).There is evidence that human and murine T cells, induced against avariety of malaria antigens, can control parasite growth in vitro and invivo. The results obtained show that HKCC can induce malariaantigen-specific T cell responses and therefore could be used as aneffective adjuvant to modulate and/or augment protective immuneresponses elicited by malaria vaccines.

Example 22: HKCC Induces Antigen Specific T Cell Responses Against M2eUpon Oral Immunization and Viral Challenge

To determine the role of HKCC as an oral adjuvant, groups of five BALB/cfemale mice were immunized twice orally (at 12 days interval) with M2elipopeptide (50 μg/mouse)+HKCC (50×10⁶ CFU/mouse), M2e lipopeptide (50μg/mouse) alone or PBS in 200 μl total volume/mouse. Twelve days afterimmunizations, mice were challenged intranasally with 30 μl/mouse ofstock of H1N1 (PR8) virus. Four days after infection mice wereeuthanized. Spleens and BALs were collected. Oral immunization with HKCCinduced antigen specific proliferation and activation of CTLs insplenocytes and enhanced infiltration of activated CTLs in BALs ascompared to M2e alone or PBS immunized groups (FIG. 22). These resultsdemonstrate that HKCC is a potent oral adjuvant and can enhance theimmunogenicity of poorly immunogenic antigens. CD8+ T cells are criticalin controlling and eliminating respiratory infections, especially thosecaused by highly pathogenic strains of influenza viruses. Importantly, Tcell responses were generated upon oral immunization of mice with M2eadmixed with HKCC in BALs and spleens of the immunized mice.

Example 23: Recombinant Adenoviral Vector Containing HCV-NS3 InducesAntigen Specific T Cell Responses

HKCC admixed with recombinant adeno-NS3 enhanced antigen specificresponses against NS3 as compared to recombinant-NS3 or PBS immunizedgroups (FIG. 23). Female C57b/6 mice (n=5/group) were immunized twice(at 14 days interval) intramuscularly with 2×10⁷ PFU/mouse adenoviralvector (rAd-NS3), rAd-NS3+HKCC, or PBS. Eight days after secondimmunization, mice were euthanized. The proliferation of spleen T cellswas determined against HCV NS3 antigen and mixture of 15-aa peptidesfrom NS3. This study demonstrates that recombinant-vector based vaccinescombined with HKCC resulted in improvement of the immunogenicity of thevector-based vaccine. These data indicate that HKCC can be used as anadjuvant with vector-based vaccines.

Examples 24: HKCC Mixed with IFA Elicits Strong T Cell ResponsesFollowing Single Subcutaneous Immunization with a Low Dose of Antigen(Vaxigrip) and Challenge with Heterologous (H1N1) Influenza Virus

Groups of five BALB/c female mice were immunized by the subcutaneousroute with

HKCC at 50×10⁶ CFU/mouse, IFA (20 μl) and Vaxigrip (0.5 μg/mouse) in 100μl total volume/mouse. In the control no adjuvant group, Vaxigrip (0.5μg/mouse) alone was administered subcutaneously. Mice were challengedwith H1N1 intranasally 8 days after immunization and euthanized 3 daysafter infection. Intriguingly, the results obtained demonstrated thatHKCC with low doses of antigen and IFA stimulates robust T cell immunity(proliferation) against seasonal flu vaccine (Vaxigrip) as compared tono adjuvant group (FIG. 24). Therefore, the HKCC adjuvanted vaxigrip(vaxigrip in an oil-in-water formulation with HKCC as an adjuvant) wassuperior to non-adjuvanted vaxigrip in inducing cellular immuneresponses after heterologous viral challenge.

Example 25: Recombinant HKCC Containing Hemagglutinin Protein fromInfluenza Virus (H5-HKCC) after Intranasal Immunization InducesInfluenza Antigens' Specific T Proliferative Responses

Groups of five BALB/c female mice were immunized with recombinantH5-HKCC or wild-type HKCC (50×10⁶ cfu/ml) twice intranasally (at 8 daysinterval) and challenged with H1N1 influenza 12 days after secondimmunization. Mice were euthanized 3 days after infection. H5-HKCCinduced influenza virus antigens' specific T cell responses, which washigher than immunization with wild-type HKCC (FIG. 25).

These results show that genetically modified HKCC expressing aheterologous polypeptide of a pathogen associated antigen (H5 ofinfluenza virus) can induce cellular immune responses against influenzaantigens upon immunization by mucosal route.

Example 26: Effect of Live CC and/or HKCC: HKCC Induces RobustAntigen-Specific Humoral Immune Responses Against Co-Administration ofMultiple Antigens of Influenza Upon Single s.c. Immunization andChallenge with Heterologous Influenza Virus

It was examined whether HKCC could be used as an efficient adjuvant withmultiple influenza antigens (M2e, and HA and NA containing Vaxigrip) toinduce antigen specific humoral immune responses against multipleantigen within a short period of time. Groups of five BALB/s female micewere immunized with a mixture of seasonal TIV influenza vaccine(Vaxigrip 1.0 μg/mouse), M2e-monolipo peptide (20 μg/mouse) and HKCC(50×10⁶ CFU/mouse); Vaxigrip (1.0 μg/mouse), M2e-monolipo peptide (20μg/mouse) and live CC (50×10⁶ CFU/mouse); Vaxigrip (1.0 μg/mouse),M2e-monolipo peptide (20 μg/mouse); or PBS once subcutaneously. Micewere challenged intranasally with H1N1 influenza virus eight days afterimmunization. Sera samples were collected 4 days after infection (11days after single immunization) and examined for antibodies againstVaxigrip and M2e (FIG. 26).

The results showed that HKCC induces early and robust antigen specificantibody (IgG, IgG1, IgG2a and IgG3) responses following singlesubcutaneous immunization against both Vaxigrip and M2e antigens, ascompared to no adjuvant or no immunization groups (FIG. 26). Incontrast, immunization of antigens with live CC led to an overallreduction in all of the antibodies measured as compared to immunizationwith antigens without adjuvant (FIG. 26). These results indicate thatcoadministration of HKCC with a licensed trivalent vaccine containing amixture of HA and NA, and a conserved antigen M2e, as a universalvaccine, induces strong antigen specific antibody responses in micewithin 11 days of immunization and heterologus virus infection.

Example 27: In Vitro Induction of IFN-α by Human PBMCs Stimulated withHKCC

TABLE 1 IFN-α (pg/ml) Saline  0 HKCC 1 × 10⁵ CFU/ml 13 (Donor #1) HKCC 1× 10⁶ CFU/ml  9 (Donor #2) HKCC 1 × 10⁷ CFU/ml  9 (Donor #3)

Human PBMCs (4×10⁶/well) were treated with HKCC (1×10⁵, 1×10⁶ and 1×10⁷CFU/ml), for 24 hours. Supernatants were collected and assayed for IFN-αby ELISA. The data are presented in Table 1. Data are representative ofthree experiments from three different individual donors. These resultsindicate that HKCC induces IFN-α response from human PBMCs. There areemerging clinical evidence that interferons are useful and viabletreatments for a variety of viral infections such as HBV, HCV,influenza, SARS, Dengue, rhinoviruses, HPV, HIV, pox viruses etc.Clinical benefits of type 1 interferons alone and in combination withchemotherapeutics have also been observed in various cancers (melanoma,renal cell carcinoma, multiple myeloma, leukemia, AIDS related Kaposi'ssarcoma). Therefore, HKCC could be used to treat various viral diseasesand cancers.

Example 28: In Vitro Induction of IL-12 by Human PBMCs Stimulated withHKCC

TABLE 2 IL-12 (pg/ml) Saline 0 HKCC 1 × 10⁵ CFU/ml 250 HKCC 1 × 10⁶CFU/ml 750 LPS (1 μg/ml) 2160

IL-12 can promote IFN-gamma production and enhance the proliferation andcytotoxicity of CTLs. It also induces an anti-angiogenic program andprovides costimulatory and anti-apoptolic signals that regulate theactivity of effector-memory T cells. Therefore, the induction of IL-12by human PBMCs upon stimulation by HKCC was examined. Human PBMCs(4×10⁶/well) were treated with HKCC (1×10⁵ and 1×10⁶ CFU/ml), for 24hours. Supernatants were collected and assayed for IL-12 by ELISA. Thedata are presented in Table 2. Data are representative of threeexperiments from three different individual donors. Results show thatHKCC is capable of activating human PBMCs in a dose dependent manner andstimulating the production of IL-12 in vitro, suggesting the activationof innate immune cells. The ability of HKCC to induce IL-12 secretion isalso a good measure of its adjuvant potential inducing TH1 immuneresponses.

Example 29: In Vitro Induction of Cytokines (pg/ml) from Human PBMCsUpon Stimulation with HKCC

TABLE 3 IFN-γ TNF-α IL-2 IL-6 IL-10 IL-17A IL-22 PBS 0.0 0.0 0.0 0.0 0.00.0 0.0 HKCC 135 41 35 1500 40 290 20 1 × 10⁶ CFU/ml HKCC 420 54 41 150066 310 199 1 × 10⁷ CFU/ml HKCC 1500 125 37 2000 183 392 228 5 × 10⁷CFU/ml

Human PBMCs (4×10⁶/well) were treated with HKCC (1×10⁶, 1×10⁷ and 5×10⁷CFU/ml), for 96 hours. Supernatants were collected and assayed forcytokines by ELISA. The data are presented in Table 3. Data arerepresentative of three experiments from three different individualdonors. These results indicate that HKCC induces regulated levels ofdiverse range multifunctional cytokines from human PBMCs, which could beproduced by various innate and/or adaptive immune cells present in PBMCsand which have been associated with protection in several diseases inhuman subjects.

Example 30: In Vivo Induction of IL-12 in Lungs Upon i.n. And OralAdministration of HKCC in Mice

TABLE 4 IL-12 (pg/ml) Saline 0 HKCC 50 × 10⁶ CFU/mouse i.n. 20 HKCC 50 ×10⁶ CFU/mouse oral 170

Recombinant IL-12 alone or combined with chemotherapy and/or monoclonalantibodies has been demonstrated to be effective in murine models andhuman clinical trials of breast cancer, metastatic melanoma, merkel cellcarcinoma, cutaneous T cell lymphoma etc. Sequential use of paclitaxeland IL-12 has been shown to reduce tumor burden in mice. To determinewhether HKCC can cause in vivo immune stimulation, HKCC or PBS wasadministered to C57bl/6 mice by intranasal and oral routes at 50×10⁶pfu/mouse dose and lung washes were collected after 5 hrs. Theproduction of IL-12 was determined by ELISA. The data are presented inTable 4. HKCC induced the production of IL-12 in lungs upon intranasaland oral administration in mice in vivo experiments. IL-12 levels werestrong and higher in mice that received HKCC orally than those receivingHKCC intranasally. Mice that received PBS had no IL-12 detected. Thisdata confirms our in vitro studies with human PBMCs and demonstratesthat HKCC by both mucosal routes activates innate immunity.

Example 31: In Vivo Induction of IFN-Beta in Serum and Lungs Upon SingleSubcutaneous, i.n. or Oral Administration of HKCC in Mice

TABLE 6 IFN-beta in serum (pg/ml) Saline 67 HKCC 50 × 10⁶ CFU/mouse s.c.127.5

TABLE 7 IFN-beta in lungs (pg/ml) Saline 20 HKCC 50 × 10⁶ CFU/mouse i.n.70 HKCC 50 × 10⁶ CFU/mouse oral 80

The ability of HKCC to induce type 1 interferon was assessed in vivo.C57bl/6 mice were administered once with HKCC (50×106 cfu/ml) by s.c.,oral and i.n. routes. Serum and lung washes were collected at 5 hrsafter HKCC administration and IFN-beta was determined by ELISA. The dataare presented in Tables 6 and 7. Significant levels of IFN-beta weredetected in the serum and lungs of mice treated with HKCC by s.c., i.n.,and oral routes, compared to PBS group. Type 1 interferons havewidespread potential as therapeutic agents for the treatment of viralinfections, microbial infections and cancers. IFN-beta was found to be apotent inhibitor of influenza and SARS-CoV (severe acute respiratorysyndrome associated with coronavirus). Interferon-beta is also usedclinically for the treatment of multiple sclerosis. These resultssuggest that HKCC is a potent IFN-beta inducer in vivo.

Example 32: In Vivo Induction of GM-CSF in Lungs Upon Single i.n.Administration of HKCC in Mice

TABLE 8 GM-CSF in lungs (pg/ml) Saline 0 HKCC 50 × 10⁶ CFU/mouse i.n. 59

The ability of HKCC to induce GM-CSF was assessed in vivo. C57bl/6 micewere administered once with HKCC (50×10⁶ cfu/ml) by i.n. route. Lungwashes were collected at 5 hrs after HKCC administration and GM-CSF wasdetermined by ELISA. The data are presented in Table 8. HKCC treatedmice had significant amount of GM-CSF in lungs compared to PBS treatedmice.

Example 33: In Vivo Induction of IL-17A in Lungs Upon Single i.n.Administration of HKCC in Mice

TABLE 9 IL-17A in lungs (pg/ml) Saline 0 HKCC 50 × 10⁶ CFU/mouse i.n. 20

IL-17A producing T helper (TH17) cells are a distinct lineage of Tcells. These cells play an important role in the host defense againstvarious pathogens. TH17 memory cells are key players in mucosalimmunity. TH17 cells play a crucial role in mounting the immunity forboth intracellular and extracellular pathogens and their primaryfunction is to clear various pathogens. Genetic deficiency in mountingan effective TH17 response in humans results in mucocutaneous andstaphylococcal lung infections. The effector CD4+ T cells defined bytheir production of IL-17A, has been found to provide protection againstbacterial, mycobacterial, fungal, and viral infections. The TH17 subsetsare deleted in chronically-HIV infected patients. Memory CD4 T cellshave a pivotal role in HIV/AIDS eradication and cure. IL-17A expressionhas also been detected in γδ T cells, NK cells, CD8+ T cells,T-follicular helper (Tfh) cells and neutrophils.

To determine whether HKCC can cause IL-17A stimulation in vivo, C57bl/6mice were administered once with HKCC (50×10⁶ cfu/ml) by i.n. route.Lung washes were collected at 5 hrs after HKCC administration and IL-17Awas determined by ELISA. The data are presented in Table 9. A markedinduction of IL-17A as observed in lungs of HKCC treated mice. Incontrast, there was no induction of IL-17A in mice treated with saline.These studies demonstrate that HKCC can induce IL-17A in an individualand therefore could be used as an immunotherapeutic to treat viraldiseases (such as HIV, HCV, HBV etc.), fungal diseases (such as C.albicans etc.), mycobacterial diseases (such as Mtb etc.), bacterialinfections (such as K. pneumonia, P. carinii, S. aureus, H. pylori, S.pneumonia, B. anthacis etc.), and parasitic diseases (such asToxoplasmosis etc.).

Example 34: In Vitro Activation of Human DCs by HKCC

TABLE 10 % positive cells CD11c CD80 CD86 DEC-205 Saline 65.5 0.3 63.77.8 HKCC 5 × 10⁷ CFU/ml 70.2 0.9 70 16.3

The effect of the HKCC on human DCs was investigated by analyzing theexpression of co-stimulatory molecules following treatment of human DCswith HKCC for 24 hrs. The data are presented in Table 10. The resultsobtained showed that HKCC induces up-regulation of expression of CD11c,CD80, CD86 and DEC-205 on human DCs.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of modulating an antigen-specific immuneresponse in an individual, the method comprising administering to theindividual an effective amount of an immunomodulatory compositioncomprising: a) heat-killed Caulobacter spp.; b) a pathogen-derivedantigen, or a tumor-associated antigen; and c) a pharmaceuticallyacceptable excipient; wherein the Caulobacter spp. is selected from thegroup consisting of Caulobacter vibroides, Caulobacter henricii,Caulobacter fusiformis, Caulobacter intermedius, and Caulobactersubvibroides, and the composition is administered via an oral, nasal,subcutaneous, intramuscular, topical or mucosal route of administration.2. The method of claim 1, wherein the immune response comprises ahumoral immune response, a cellular immune response, an innate immuneresponse, and/or production of one or more cytokines.
 3. The method ofclaim 1, further comprising administering to the individual a cancerchemotherapeutic agent, an anti-bacterial agent, an anti-mycobacterialagent, an anti-viral agent, an anti-protozoan agent, an anti-malarialagent, an anti-helminth agent, or a therapeutic treatment for cancerselected from the group consisting of radiation therapy, laser therapy,photodynamic therapy, and surgery.
 4. The method of claim 1, furthercomprising administering to the individual an antibody.
 5. The method ofclaim 1, further comprising administering to the individual a cytokine,an adjuvant, or an immunomodulatory agent.
 6. The method of claim 1,wherein the individual is a human, a non-human mammal, or a non-mammalanimal.
 7. A method of reducing tumor progression or an infection in anindividual having a disease selected from the group consisting of cancerand an infectious disease, wherein the infectious disease is caused by avirus, a bacterium, or a fungus, the method comprising administering tothe individual a composition comprising: a) heat-killed Caulobacterspp.; and b) a pharmaceutically acceptable excipient; wherein theCaulobacter spp. is selected from the group consisting of Caulobactervibroides, Caulobacter henricii, Caulobacter fusiformis, Caulobacterintermedius, and Caulobacter subvibroides, and the composition isadministered via an oral, nasal, subcutaneous, intramuscular, topical ormucosal route of administration.
 8. The method of claim 7, furthercomprising administering an anti-bacterial agent, an anti-mycobacterialagent, an anti-viral agent, an anti-protozoan agent, an anti-malarialagent, an anti-helminth agent, an immunomodulatory agent, an antibody, acytokine, an adjuvant, a vaccine, a cancer chemotherapeutic agent, or atherapeutic treatment for cancer selected from the group consisting ofradiation therapy, laser therapy, photodynamic therapy, and surgery. 9.The method of claim 7, wherein the individual is a human, a non-humanmammal, or a non-mammal animal.
 10. A method of enhancing the efficacyand/or reducing the toxicity of an anticancer therapeutic treatment inan individual, the method comprising administering to the individual aneffective amount of an immunomodulatory composition comprising: a)heat-killed Caulobacter spp.; and b) a pharmaceutically acceptableexcipient; wherein the Caulobacter spp. is selected from the groupconsisting of Caulobacter vibroides, Caulobacter henricii, Caulobacterfusiformis, Caulobacter intermedius, and Caulobacter subvibroides, andthe composition is administered via an oral, nasal, subcutaneous,intramuscular, topical or mucosal route of administration.